Tetraploid Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) Rootstocks Improve Common Clementine Tolerance to Long-term Nutrient Deficiency (Citrus clementina Hort. ex Tan)

Tetraploid Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) Rootstocks Improve Common Clementine Tolerance to Long-term Nutrient Deficiency (Citrus clementina Hort. ex Tan) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tetraploid Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) Rootstocks Improve Common Clementine Tolerance to Long-term Nutrient Deficiency (Citrus clementina Hort. ex Tan) Julie Oustric, Stéphane Herbette, Yann Quilichini, Raphaël Morillon, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-116997/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Apr, 2021 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Nutrient deficiency alters growth and the production of high-quality nutritious food. In Citrus crops, rootstock technologies have become a key tool for enhancing tolerance to abiotic stress. The use of doubled diploid rootstocks can improve adaptation to lower nutrient inputs. This study investigated leaf structure and ultrastructure and physiological and biochemical parameters of common clementine scions (C) grafted on diploid (2x) and doubled diploid (4x) Carrizo citrange (C/CC2x and C/CC4x) and Citrumelo 4475 (C/CM2x and C/CM4x) rootstocks under optimal fertigation and after seven months of nutrient deficiency. Rootstock ploidy level had no impact on structure but induced changes in the number and/or size of cells and some cell components of common clementine leaves under optimal nutrition. Rootstock ploidy level did not modify gas exchanges in Carrizo citrange but induced a reduction in the leaf net photosynthetic rate in Citrumelo 4475. By assessing foliar damage, changes in photosynthetic processes and malondialdehyde accumulation, we found that C/CM4x were less affected by nutrient deficiency than the other scion/rootstock combinations. Their greater tolerance to nutrient deficiency was probably due to the better performance of the enzyme-based antioxidant system. Nutrient deficiency had similar impacts on C/CC2x and C/CC4x. Tolerance to nutrient deficiency can therefore be improved by rootstock polyploidy but remains dependent on the rootstock genotype. Space Exploration Mechanical Engineering Nutrient deficiency nutritious food Citrus crops genotype Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Fruit crops, especially citrus fruits, require large amounts of fertilizers to ensure good production and fruit quality. Today, the challenge for sustainable agriculture, and particularly organic agriculture, is to reduce the use of inputs in crops. Reducing inputs optimizes the economic outcome while limiting the environmental impact. Water and minerals absorbed by roots in the soil are essential for plant development, growth and reproduction. Fourteen minerals are considered essential and these can be divided into two groups: the macroelements (N, K, P, Ca, Mg and S) which are constituents of organic matter (proteins, nucleic acids) or play a strong osmotic role, and microelements (Zn, Cu, Fe, Mn, B, Mo, Cl and Ni), which are only involved as specific cofactors or constituents of certain enzymes 1–3 . Mineral-deficient plants present various visual symptoms, such as necrosis, chlorosis, dark green foliage, or stunted growth 4 . Essential mineral deficiency alters plant primary metabolism and this disrupts the physiological and biochemical processes leading to changes in leaf structure and ultrastructure 5,6 . Moreover, cells must cope with an overproduction of reactive oxygen species (ROS) such as singlet oxygen (O * ), hydroxyl radicals (OH • ), superoxide anion (O 2 • − ) and hydrogen peroxide (H 2 O 2 ) which cause membrane leakage due to lipid peroxidation and damage to proteins and nucleic acids 7–9 . As a result, ROS defence mechanisms are activated by a set of antioxidant compounds (metabolites such as ascorbate and proline) and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR)). Adapted genotypes are therefore sought in order to propose cultural itineraries more suitable to low input conditions. In citrus crops, improving the performance of varieties is based on the scion/rootstock combination forming the aerial parts and roots of the plant, respectively. The impact of the rootstock on the scion lies in its influence on flowering, fruit quality, canopy size, and resistance, among other parameters 10 . Good relations between rootstock and scion are essential for the efficient translocation of water and mineral nutrients and to promote biomass production and tolerance to biotic and abiotic factors, such as nutrient deficiency 11,12 . Improved root system vigour in citrus rootstocks results in increased soil nutrient and water uptake 13,14 . All modern cultivated varieties of citrus are now grafted on diploid rootstocks and therefore have two sets of chromosomes in their genetic heritage. However, incomplete mitosis of somatic embryos may occur in seedlings of diploid (2x) apomictic genotypes with formation of doubled diploid (4x) genotypes 15 . Recent advances have shown that rootstock tetraploidy can improve the tolerance of a scion. Grafting of Valencia Delta sweet orange ( Citrus sinensis L.) on 4x rangpur lime ( Citrus limonia ) rootstock was found to improve its tolerance to water stress by changing patterns of gene expression in Rangpur lime citrus roots regulating adaptation to water deficit 16 . Natural chilling stress tolerance associated with a robust antioxidant system was also enhanced in common clementine ( Citrus clementina Hort. ex Tan) grafted with Carrizo citrange ( Citrus sinensis Osb.× Poncirus trifoliata L. Raf.) 4x rootstock 17 . Chromium tolerance of Kinnow mandarin ( Citrus nobilis Lour x Citrus deliciosa Ten) grafted on three 4x rootstocks (Poncirus trifoliata [L.], Citrus reshni, and Citrus limonia Osbeck.) may be attributed to chromium sequestration in roots with lower transfer to leaves in 4x rootstocks 18 . The use of rootstocks better adapted to environmental constraints seems to be a promising eco-friendly strategy. Many Citrus genotypes are used as rootstock for citrus cultivation. Genotypes belong either to the Citrus genus such as Volkamer lemon or are obtained by hybridization between Citrus and Poncirus genus progenitors such as Citrumelo 4475 and Carrizo citrange. Volkamer lemon which is used as rootstock for lemon, is adapted to dry, calcareous and saline soils and presents tolerance to Tristeza, cachexia and exocortis. Citrumelo 4475 imparts cold tolerance to the scion. Citrange Carrizo is frequently used in acidic and neutral soils but not in dry areas because of its limited performance under drought conditions. These two genotypes inherited Tristeza tolerance from their Trifoliate orange progenitor and give clementine varieties that produce a high yield and fruit quality 19 . In a previous study, we compared leaf structure and ultrastructure under nutrient deficiency of two genotypes used worldwide as rootstock for citrus cultivation, the Citrumelo 4475 ( Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) and Volkamer lemon ( Citrus limonia Osb.) with both 2x and 4x genotypes 20 . Results showed an increase in tolerance to nutrient deficiency in 4x genotypes. Doubled diploid genotypes presented less degradation of ultrastructural components such as chloroplasts, thylakoids, mitochondria and starch grains associated with a smaller decrease in leaf net photosynthetic rate ( P net ), stomatal conductance ( g s ) and chlorophyll fluorescence ( F v / F m ) compared to 2x genotypes. The higher tolerance to nutrient deficiency was linked to the reduced accumulation of malondialdehyde (MDA) and H 2 O 2 in Citrumelo 4475 4x than in the 2x, implying a more efficient antioxidant system in the 4x genotype. However, few differences in the antioxidant system and oxidative status were observed between 2x and 4x Volkamer lemons. The aim of this study was therefore to determine the impact of rootstock ploidy level on the 2x common clementine scion during nutrient deficiency. We selected common clementine grafted on two rootstocks used worldwide for clementine cultivation, i.e. Citrumelo 4475 (C/CM2x) and Carrizo citrange (C/CC2x) in both 2x and 4x types (C/CM4x and C/CC4x, respectively). The effect of rootstock ploidy level on common clementine scions under nutrient deficiency was investigated by studying common clementine leaf structure and ultrastructure and physiological and biochemical parameters. Results And Discussion Effect of rootstock ploidy level on anatomical properties of common clementines under control conditions Whatever the rootstock genotype and ploidy level, microscopic examination of leaf surface imprints confirmed the presence of stomata only on their abaxial surface that were surrounded by ordinary epidermal cells 29 (anomocytic organization) (Fig. 1). Rootstock tetraploidy did not induce any changes in the location of stomata or the epidermal cell structure of common clementine leaves. This agrees with this anomocytic organization observed on leaves of tetraploid seedlings 20 . Stomata size was unchanged in C/CM4x and C/CC4x combinations. Conversely, stomata density decreased and ostiole sizes increased in C/CC4x compared to C/CC2x combinations (Table 1). Studies have shown a positive correlation between stomata and ostiole size and a negative correlation between stomata density and ploidy level in both 4x ungrafted genotypes and 3x clementine in comparison to their 2x counterparts 20,29,30 . Our study showed that 4x rootstocks had no effect on stomata sizes in 2x common clementine scions and a non-systematic impact on ostiole size and stomatal density, putatively through changes in hydraulics or mineral inputs. The decrease in stomatal density caused by rootstock tetraploidy in the C/CC4x combination was not associated with any changes in gas exchanges compared to its C/CC2x counterpart (Table 1; Fig. 1 and 5). The increase in ostiole size in the C/CC4x combination compared to its C/CC2x counterpart suggests an adjustment of the stomata opening to compensate for the reduced stomatal density and maintain stomatal conductance 31 . This adjustment could be due to the considerable degradation of starch grains in the guard cells in the first hour of light contributing to a rapid increase in the opening of the stomata in parallel with the activation of membrane ion transport 32,33 . Despite identical physical stomatal attributes between C/CM2x and C/CM4x combinations, analysis of the gas exchange parameters revealed a decrease of P net in the C/CM4x combination (Table 1; Fig. 1 and 5). Other factors than leaf structure therefore appear to be involved in the regulation of photosynthesis in the C/CM4x combination. At ultrastructural level, rootstock tetraploidy initiated changes in the number and size of the main cell components of the 2x common clementine leaves (Tables 2 and 3). Cell size and thickness in both palisade and spongy mesophylls were reduced in C/CC4x compared to C/CC2x, whereas they increased in C/CM4x compared to C/CM2x (Tables 2 and 3; Fig. 2 and 3). These ultrastructural modifications were the only identical changes in C/CM4x and the ungrafted rootstock counterpart 20 . In contrast to its ungrafted counterpart, C/CM4x showed a lower P net and similar chloroplast numbers to C/CM2x 20 (Tables 2 and 3; Fig. 2, 3 and 5A). Chloroplast enlargement in palisade mesophyll brought about by rootstock tetraploidy in common clementine leaves was not associated with an increase in chloroplast numbers. This phenomenon appears to reduce photosynthetic capacity 34 . Overall, under optimal conditions and depending on the rootstock genotypes, the structural and/or ultrastructural modifications of common clementine leaves induced by rootstock tetraploidy could either compensate each other with no change in gas exchanges or induce a modification of gas exchanges. The number and size of transitory starch grains and mitochondria were either reduced or similar between 2x and 4x scion/rootstock combinations in palisade and spongy mesophylls (Tables 2 and 3; Fig. 2 and 3). The number of plastoglobuli increased in C/CM4x and C/CC4x compared to C/CM2x and C/CC2x in the chloroplasts of the palisade and spongy mesophylls. Only the C/CM4x plastoglobuli were larger than those of its 2x counterpart (Tables 2 and 3; Fig. 2 and 3). Thus, rootstock tetraploidy involved a potential increase in lipid (plastoquinone-9 (PQ-9), plastoquinol-9 (PQ-9H2) and a-tocopherol (a-T)) reserves that could not be deposited in the thylakoids of chloroplasts. Rootstock tetraploidy can induced modifications in the number and/or size of the investigated ultrastructural components. Differences in photosynthetic properties and redox status of scion/rootstock combinations under nutrient stress could be related to their leaf and cell anatomy Complete starvation resulted in a significant decrease in N but similar or higher levels of P, K, Mg, Ca and Na than controls in all scion/rootstock combinations (Table 4). A concentration effect induced by the transfer of N to other tree areas could explain the increase in P, K and Mg contents in all scion/rootstock combinations 35 . Leaf chlorosis occurs when plants do not have the nutrients needed for chlorophyll synthesis, which in turn affects the photosynthetic efficiency 36 . However, the decrease in N, which was an important factor in chlorosis, was similar in all scion/rootstock combinations (Table 4). C/CM4x showed less chlorosis as indicated by its light green colour compared with the yellow colour of other scion/rootstock combinations (Fig. 4). These results suggest a better integrity of chlorophyll content probably related to a better protection against ROS 37–39 . The improvements in the redox status of scions by tetraploid rootstocks is probably linked to an improvement in photosynthesis. Chlorosis was associated with structural and ultrastructural foliar changes and a disruption of photosynthetic properties in all scion/rootstock combinations 6,40 (Tables 1, 2 and 3; Fig. 5). At structural level, stomata and ostioles of all scion/rootstock combinations showed a narrowing which was associated with an increase in density (except in C/CC2x) response to nutrient deficiency (Table 1; Fig. 1). The rootstock apparently has a different effect on the structural components of common clementine leaves depending on genotype and/or ploidy level. P net and g s decreased concomitantly in all scion/rootstock combinations under nutrient deficiency (Fig. 5A-B). These results suggest a critical role of stomatal structure in the process of CO 2 availability 41 . However, the smaller decrease in P net , g s and F v / F m in C/CM4x than in other scion/rootstock combinations (Fig. 5) implies that other factors than stomata are needed to sustain photosynthesis 42–44 . At ultrastructural level, nutrient deficiency resulted in cell size enlargement (except in C/CM2x spongy mesophyll), thylakoid with grana degradation and a decrease in chloroplast size in leaf mesophylls of all scion/rootstock combinations (Tables 2 and 3; Fig. 2 and 3). Similar results were recorded in other plants under conditions of high light stress, infection, dark-induced senescence or total nutrient deficiency 6,20,45,46 . Chloroplast degradation may be due to the significant decrease in N in the mature leaves of each scion/rootstock combination following the extensive remobilization of N present in the cell to younger leaves or storage areas during nutrient deficiency 47,48 (Tables 2 and 3; Fig. 2 and 3). The overproduction of reactive oxygen species (ROS) leads to alterations in the integrity of the membrane structures of cells, chloroplasts or thylakoids by a process called lipid peroxidation, one of the indicators of which is malondialdehyde (MDA). The lower MDA content in C/CM4x compared to other scion/rootstock combinations was consistent with the reduced damage to ultrastructure (Tables 2 and 3; Fig. 2, 3, and 6D). Thylakoid size and structure is dependent on the formation of PSII-LHCII supercomplexes 49 . In C/CM4x, the lower decrease in F v / F m suggests a more limited degradation of the PSII-LHCII supercomplexes reducing the disruption of electron transport for the photosynthetic reaction and thus the production of ROS 50 (Fig. 5C and 6D) . Maintaining the redox balance in C/CM4x decelerates the degradation of thylakoids resulting in a more efficient photosynthetic capacity ( P net and F v / F m ) compared to C/CM2x, C/CC2x and C/CC4x after nutrient deficiency (Tables 2 and 3; Fig. 2, 3, 5A, 5C and 6D). Plastoglobuli are bound to thylakoids via their membranes. Studies have shown that plastoglobuli are involved in the formation and degradation of thylakoids during plant growth, development and senescence but also when plants are exposed to drought, high-light, N starvation or fungal infection 46,51–53 . The reduced damage of the thylakoid membrane in C/CM4x would explain the maintenance of the plastoglobuli numbers and the small increase in their size due to the accumulation of catabolites formed by thylakoid degradation in their hydrophobic core compared to other scion/rootstock combinations 54,55 (Tables 2 and 3; Fig. 2 and 3). Rootstock tetraploidy improves the tolerance of 2x common clementine scions by maintaining the redox status and delaying ultrastructural changes and damage with a consequent improvement in photosynthetic capacity. However, as suggested by the similar impact of nutrient deficiency on C/CC2x and C/CC4x, rootstock tetraploidy does not automatically result in better tolerance of the photosynthetic properties of the scion. Differences in tolerance to nutrient stress between scion/rootstock combinations may be explained by a better antioxidant system Nutrient deprivation led to a modification of oxidative metabolism 35,39,56 . Tolerance differences between scion/rootstock combinations may be related to differences in ROS production and/or antioxidant system efficiency. The enzymatic antioxidant system response differs according to plant species and the deficient minerals 56–60 . Enzymatic antioxidant systems in common clementine leaves differed depending on the rootstock genotypes (Fig. 6). Nutrient deficiency impaired SOD activity in C/CC4x and C/CM4x (Fig. 6A). It was interesting to note that APX activity either increased or remained similar to the control in all scion/rootstock combinations (Fig. 6C) whereas CAT activity only increased in C/CM4x under nutrient deficiency (Fig. 6B) . Concurrent CAT and APX activity is important for the elimination of H 2 O 2 61–63 . In C/CM2x, C/CC2x and C/CC4x, the low H 2 O 2 and high MDA contents were due to increased OH • formation (Fig. 6D and E). This OH • is either directly formed by the addition of electrons to the O 2 •− not transformed into H 2 O 2 by SOD and/or by the transformation of H 2 O 2 via Fenton or Haber-Weiss reactions when the APX activity is insufficient to compensate for the low CAT activity. Conversely, in C/CM4x, the synergistic activity of CAT and APX maintains the MDA content in C/CM4x (Fig. 6B, C and D). As in their ungrafted counterparts, the increased enzymatic activity may explain the reduced ultrastructural damage and decrease in photosynthetic activity in C/CM4x in comparison with the other scion/rootstock combinations. The improvement in performance of the enzyme-based antioxidant system in the scion is due to the tetraploidization of the rootstock and the compatibility between rootstock and scion. In conclusion, rootstock ploidy level had no effect on the structure of the common clementine scion leaves (except stoma density in Carrizo citrange) whereas it induced modifications in the ultrastructural components. The impact of prolonged nutrient deficiency on the structure, ultrastructure, physiology and biochemistry of the common clementine scion differed according to the variety and ploidy level of the rootstock. Among the four scion/rootstock combinations, common clementine grafted with 4x citrumelo 4475 rootstock (C/CM4x) was the most tolerant to nutrient deficiency as indicated by the limited changes in leaf cell structures and photosynthetic activity. The improved tolerance of common clementine grafted with 4x citrumelo 4475 rootstock may be related to a better antioxidant system. Tolerance to nutrient deficiency can therefore be improved by rootstock polyploidy but remains dependent on the rootstock genotype. The next step of this study will be to test the impact of rootstock ploidy level on the quality and yield of clementine fruit under nutrient deficiency. Materials And Methods Plant material and experimental design The experiment was carried out on the AREFLEC experimental station located in San Giuliano, Corsica (41° 47' 27’’N and 09° 23' 40’’E). 2x common clementine ( Citrus clementina Hort. ex Tan; SRA 92) scion grafted onto one year seedlings of Carrizo citrange ( Citrus sinensis L. Osb . × Poncirus trifoliata L. Raf.) and Citrumelo 4475 ( Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) 2x (C/CC2x and C/CM2x, respectively) and their 4x counterparts (C/CC4x and C/CM4x, respectively) were used as source materials. The ploidy status of six seedlings for each combination was first checked by flow 10 cytometry (Partec I, Germany) according to Froelicher et al. 21 . Clonal propagation by nucellar embryogenesis was checked by genotyping using SSR markers as described in Vieira et al. 22 . The 24 selected scion/rootstock combinations were then grown under identical conditions in vermiculite with fertigation and water (1L/h) for three years in a tunnel greenhouse. The stock solution used for irrigation included: 20-5-10 NPK + 2MgO fertilizer + trace elements according to the recommendations of the French department of agriculture. Seedlings were divided into two blocks: one with reference fertigation (control plants) and the other with irrigation water (without nutrient inputs). A total of three plants of each scion/rootstock combination were randomized by fertigation level ( n = 3). The fertigation solutions were prepared and applied with a metering pump. Before starting the experiment, the vermiculite was washed for 48 hours in order to eliminate any nutritional reserves in the pot. According to a previous experiment of Oustric et al. 20 , leaf and root samples were collected and physiological measurements made from May to December 2018 at two different times (days): 0 (D0: control plant) and 210 (D210) days after the start of nutritional deprivation. Measurements were made and samples taken from homogeneous plants comprising four branches with fully-expanded leaves developed under stress and control conditions. Scanning electron microscopy (SEM) Scanning electron microscopy measurements were carried out on three leaf pieces per scion/rootstock combination and fertigation level (typically 1 cm²) ( n = 3) cut with a razor blade from mid-laminar areas at between 10:00 and 11:00 am. As described in Oustric et al. 20 , leaves were then immediately fixed in cold (4 °C) 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in a 0.1 M cacodylate buffer at pH 7.2, dehydrated through a graded ethanol series (30%, 50%, 75%, 90% and 100%) and dried under CO 2 in an Emitech K850 critical point dryer (Quorum Technologies Ltd, Ashford, U.K.). Specimens were mounted on aluminum stubs with carbon double-sided adhesive disks, coated with gold/palladium in a SC7640 sputter coater (Quorum Technologies Ltd, Newhaven, U.K.) and examined under a S-3400N scanning electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) at an accelerating voltage of 5 kV. Transmission electron microscopy (TEM) Transmission electron microscopy measurements were carried out on five leaf pieces per scion/rootstock combination and fertigation level (typically 1 mm²) ( n = 5) cut with a razor blade from mid-laminar areas at between 10:00 and 11:00 am. As described in Oustric et al. 20 , leaves were immediately fixed in cold (4 °C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in a 0.1 M cacodylate buffer at pH 7.2, post-fixed in cold (4 °C) 1% osmium tetroxide in the same buffer for 1 h, dehydrated through a graded ethanol series (70% and 100%) and propylene oxide, embedded in Spurr, and polymerized at 60 °C for 24 h. Ultra-thin sections (60–90 nm) were cut using a Power tome PC ultramicrotome (RMC Boeckeler, Tuscon, U.S.A.). Sections were placed on 200- and 300-mesh copper grids and stained with UranyLess (Delta Micoscopies, France) and lead citrate. They were then examined using a Hitachi H-7650 (Hitachi High-Technologies Corporation, Tokyo, Japan) at an accelerating voltage of 80 kV. Mineral content was measured on a pool of eight fully expanded leaves for the three plants per combination and fertigation level ( n = 3) between 10:00 and 11:00 am. Fresh leaves were placed in a forced air oven at 65 ± 10° C overnight and then transferred into a desiccator for cooling. The dehydrated leaves were then sent to the CIRAD “Analyses des eaux, sols et végétaux service unit” at Montpellier (France) for analysis of macro- and micro-nutrients. Leaf P, K, Ca, Mg and Na contents were measured using an Agilent 720 simultaneous ICP-OES after double calcination with silica removal by adding hydrofluoric acid. The leaf total N content was evaluated after combustion using a Leco TruMac N determinator. Measurements of gas exchange and chlorophyll a fluorescence All measurements were made on three fully developed leaves for each of the three plants per combination and fertigation level ( n = 9). A portable photosynthesis system (LI600) was used to measure the leaf net photosynthetic rate ( P net ), stomatal conductance ( g s ) and transpiration rate (E) at between 7:00 and 11:00 am. The carbon dioxide concentration (CO 2 ), airflow rate, light intensity and temperature of the leaf chamber were maintained at 380 μmol.mol -1 , 500 μmol.s -1 , 1400 μmol.m -2 .s -1 and 25 °C, respectively. Chlorophyll a fluorescence parameters were measured using an OS1p (Hansatech, Instruments Ltd) at between 9:00 and 11:00 am. Leaves were dark-acclimated for 30 min using special leaf clips. Chlorophyll a fluorescence was recorded after illumination with red actinic light (650nm, 3000 μmol photon.m -2 .s -1 ) for 1 s and this was used to calculate the maximum fluorescence [ F v / F m = ( F m – F o )/F m ] 23 . Leaves were exposed to an actinic light to evaluate the current fluorescence yield ( F s ) and the actual light-adapted fluorescence ( F m ′). Formulas were applied to this data in order to determine the effective quantum yield of PSII Y(II) = ( F m ’− F s )/ F m ’], the Y(NO) = F s / F m ], the non-photochemical quenching coefficient [Y(NPQ) = ( F s / F m ’)-Y(NO)] 24,25 , and the electron transport rate through PSII [ETR(II) = Y(II) x PAR x 0.5 x 0.84] 26 . The ETR/ P net ratio was calculated to estimate the use of electrons in other processes unrelated to the photosynthetic CO 2 assimilation rate. Determination of oxidative stress and antioxidant levels Biochemical analyses were performed on three samples for each scion/rootstock combination, i.e. one per tree, obtained by pooling eight fully-expanded leaves ( n = 3) collected between 10:00 and 11:00 am and immediately immersed in liquid nitrogen and stored at -80 °C. Immediately prior to biochemical analysis, each leaf and root sample was ground to a fine powder in liquid nitrogen. Malondialdehyde (MDA) and antioxidant enzyme activities (SOD, CAT, and APX) were assayed as defined by Santini et al. 27 Hydrogen peroxide (H 2 O 2 ) was assayed using the PeroxiDetect kit (Sigma-Aldrich). This technique is based on the oxidation of ferrous (Fe 2+ ) to ferric ions (Fe 3+ ) by hydroperoxides which react with xylenol orange (“3,3′-bis[N,N-bis(carboxymethyl)aminomethyl] o-cresolsulfonephthalein, sodium salt”) to form a blue complex visible at 560 nm. Proline content was measured as described by Oustric et al. 28 A V-630 spectrophotometer was used for all measurements (Jasco Inc., Tokyo, Japan). Statistical analyses All statistical measurements were performed with R statistical software (v.2.12.1) ( http://www.R-project.org ) and the Rcmdr package. The qualitative factors studied were sampling date (D0 and D210 after nutrient deficiency), the comment clementine scion grafted onto rootstocks subjected to nutrient stress (C/CC and C/CM) and the ploidy level of nutrient stressed rootstocks (C/CC2x, C/CC4x, C/CM2x and C/CM4x). The influence of these three factors was analyzed using a two-way ANOVA followed by LSD test at p < 0.05. The microscopic data obtained on leaves of common clementine scion grafted onto the various rootstocks at D0 (control) and D210 of nutrient deficiency were analyzed by heatmaps generated by Heatmap.2 function of the gplot package 3.0.1 for Rstudio (v.1.3.1093) (https://rstudio.com). Declarations Acknowledgements This work was funded by the “Collectivité de Corse” as part of the “Innov’agrumes” research project (FEDER). We thank Jean-Claude Ribaut for hosting this study at the AREFLEC experimental station, and Gilles Paolacci and Paul Martin for installing and monitoring the fertigation system in the greenhouse. Author information Affiliations Laboratoire Biochimie et Biologie Moléculaire du Végétal, CNRS, UMR 6134 SPE, Université de Corse, Corte, France Julie Oustric, Jean Giannettini, Liliane Berti & Jérémie Santini UCA, INRA, PIAF, Clermont-Ferrand, France Stéphane Herbette Laboratoire Parasites et Ecosystèmes Méditerranéens", CNRS, UMR 6134 SPE, Université de Corse, Corte, France Yann Quilichini Equipe "Amélioration des Plantes à Multiplication Végétative", UMR AGAP, Département BIOS, CIRAD, Station de Roujol, Petit-Bourg, Guadeloupe Raphael Morillon Contributions O.J. collected data, did the statistical analysis, interpreted the results and drafted the manuscript. S.J. designed the study and drafted the manuscript. Q.Y. participated collect microscopic data. M.R., H.S., G.J. and B.L. designed the study and helped draft the manuscript. Ethics declarations Competing interests The authors declare no competing interests. References Marschner, H. Mineral nutrition of higher plants 2nd edition. Acad. G. B. (1995). Grusak, M. A. Plant Macro-and Micronutrient Minerals. E LS (2001). Lal, R. Enhancing crop yields in the developing countries through restoration of the soil organic carbon pool in agricultural lands. Land Degrad. Dev. 17 , 197–209 (2006). Ericsson, T. Growth and shoot: root ratio of seedlings in relation to nutrient availability. in Nutrient uptake and cycling in forest ecosystems 205–214 (Springer, 1995). Kalaji, H. M. et al. Identification of nutrient deficiency in maize and tomato plants by in vivo chlorophyll a fluorescence measurements. Plant Physiol. Biochem. 81 , 16–25 (2014). Du, Q. et al. Effects of potassium deficiency on photosynthesis, chloroplast ultrastructure, ROS, and antioxidant activities in maize ( Zea mays L.). J. Integr. Agric. 18 , 395–406 (2019). Mittler, R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7 , 405–410 (2002). Mittler, R., Vanderauwera, S., Gollery, M. & Van Breusegem, F. Reactive oxygen gene network of plants. Trends Plant Sci. 9 , 490–498 (2004). Apel, K. & Hirt, H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55 , 373–399 (2004). Kucukyumuk, Z. & Erdal, I. Rootstock and cultivar effect on mineral nutrition, seasonal nutrient variation and correlations among leaf, flower and fruit nutrient concentrations in apple trees. Bulg. J. Agric. Sci. 17 , 633–641 (2011). Martínez-Ballesta, M. C., Alcaraz-López, C., Muries, B., Mota-Cadenas, C. & Carvajal, M. Physiological aspects of rootstock–scion interactions. Sci. Hortic. 127 , 112–118 (2010). Schwarz, D., Rouphael, Y., Colla, G. & Venema, J. H. Grafting as a tool to improve tolerance of vegetables to abiotic stresses: Thermal stress, water stress and organic pollutants. Sci. Hortic. 127 , 162–171 (2010). Taylor, B. K. & Dimsey, R. T. Rootstock and scion effects on the leaf nutrient composition of citrus trees. Aust. J. Exp. Agric. 33 , 363–371 (1993). Lu, Z.-J. et al. The effects of inarching Citrus reticulata Blanco var. tangerine on the tree vigor, nutrient status and fruit quality of Citrus sinensis Osbeck ‘Newhall’trees that have Poncirus trifoliata (L.) Raf. as rootstocks. Sci. Hortic. 256 , 108600 (2019). Cameron, J. Genetics, breeding and nucellar embryony. Citrus Ind. 325–370 (1968). Allario, T. et al. Tetraploid Rangpur lime rootstock increases drought tolerance via enhanced constitutive root abscisic acid production. vol. 36 (2013). Oustric, J. et al. Tetraploid Carrizo citrange rootstock ( Citrus sinensis Osb. × Poncirus trifoliata L. Raf.) enhances natural chilling stress tolerance of common clementine ( Citrus clementina Hort. Ex Tan J. Plant Physiol. 214 , 108–115 (2017). Balal, R. M. et al. Kinnow mandarin plants grafted on tetraploid rootstocks are more tolerant to Cr-toxicity than those grafted on its diploids one. Environ. Exp. Bot. 140 , 8–18 (2017). Jacquemond, C., Curk, F. & Heuzet, M. Les clémentiniers et autres petits agrumes . (Editions Quae, 2013). Oustric, J. et al. Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiol. Biochem. 135 , 372–384 (2019). Froelicher, Y. et al. Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets. Plant Cell Rep. 26 , 937–944 (2007). Vieira, D. D. S. S. et al. Polyploidization alters constitutive content of volatile organic compounds (VOC) and improves membrane stability under water deficit in Volkamer lemon ( Citrus limonia Osb.) leaves. Environ. Exp. Bot. 126 , 1–9 (2016). Maxwell, K. & Johnson, G. N. Chlorophyll fluorescence—a practical guide. J. Exp. Bot. 51 , 659–668 (2000). Kramer, D. M., Johnson, G., Kiirats, O. & Edwards, G. E. New fluorescence parameters for the determination of Q A redox state and excitation energy fluxes. Photosynth. Res. 79 , 209 (2004). Klughammer, C. & Schreiber, U. Complementary PS II quantum yields calculated from simple fluorescence parameters measured by PAM fluorometry and the Saturation Pulse method. PAM Appl. Notes 1 , 201–247 (2008). Bilger, W., Schreiber, U. & Bock, M. Determination of the quantum efficiency of photosystem II and of non-photochemical quenching of chlorophyll fluorescence in the field. Oecologia 102 , 425–432 (1995). Santini, J. et al. Comparison of photosynthesis and antioxidant performance of several Citrus and Fortunella species (Rutaceae) under natural chilling stress. Trees 27 , 71–83 (2013). Oustric, J. et al. Nutrient deficiency tolerance in citrus is dependent on genotype or ploidy level. Front. Plant Sci. 10 , 127 (2019). Padoan, D., Mossad, A., Chiancone, B., Germana, M. A. & Khan, P. S. S. V. Ploidy levels in Citrus clementine affects leaf morphology, stomatal density and water content. Theor. Exp. Plant Physiol. 25 , 283–290 (2013). Jaskani, M. J., Khan, M. M. & Khan, I. A. Growth, morphology and fruit comparison of diploid and tetraploid Kinnow mandarin. Pak. J. Agric. Sci. 39 , 126–128 (2002). Büssis, D., von Groll, U., Fisahn, J. & Altmann, T. Stomatal aperture can compensate altered stomatal density in Arabidopsis thaliana at growth light conditions. Funct. Plant Biol. 33 , 1037–1043 (2006). Sack, F. D. & Paolillo Jr, D. J. Protoplasmic changes during stomatal development in Funaria. Can. J. Bot. 61 , 2515–2526 (1983). Horrer, D. et al. Blue light induces a distinct starch degradation pathway in guard cells for stomatal opening. Curr. Biol. 26 , 362–370 (2016). Okie, J. G., Smith, V. H. & Martin-Cereceda, M. Major evolutionary transitions of life, metabolic scaling and the number and size of mitochondria and chloroplasts. Proc. R. Soc. B Biol. Sci. 283 , 20160611 (2016). Tewari, R. K., Kumar, P. & Sharma, P. N. Oxidative stress and antioxidant responses in young leaves of mulberry plants grown under nitrogen, phosphorus or potassium deficiency. J. Integr. Plant Biol. 49 , 313–322 (2007). Sárvári, É. Effect of Cd on the iron re-supply-induced formation of chlorophyll-protein complexes in cucumber. Acta Biol. Szeged. 52 , 183–186 (2008). Shimshi, D. Leaf chlorosis and stomatal aperture. New Phytol. 66 , 455–461 (1967). Rodriguez de Cianzio, S., Fehr, de W. & Anderson, I. C. Genotypic Evaluation for Iron Deficiency Chlorosis in Soybeans by Visual Scores and Chlorophyll Concentration 1. Crop Sci. 19 , 644–646 (1979). Cakmak, S., Gülüt, K. Y., Marschner, H. & Graham, R. D. Effect of zinc and iron deficiency on phytos1derophore release in wheat genotypes differing in zinc efficiency. J. Plant Nutr. 17 , 1–17 (1994). Wu, X., Lu, X., Riaz, M., Yan, L. & Jiang, C. Boron toxicity induced specific changes of cell ultrastructure and architecture of components in leaf center and tip of trifoliate orange [Poncirus trifoliata (L.) Raf.]. J. Environ. Manage. 246 , 426–433 (2019). Flexas, J. et al. Diffusional conductances to CO2 as a target for increasing photosynthesis and photosynthetic water-use efficiency. Photosynth. Res. 117 , 45–59 (2013). Epron, D. & Dreyer, E. Stomatal and non stomatal limitation of photosynthesis by leaf water deficits in three oak species: a comparison of gas exchange and chlorophyll a fluorescence data. Ann. Sci. For. 47 , 435–450 (1990). Flexas, J. & Medrano, H. Drought‐inhibition of Photosynthesis in C3 Plants: Stomatal and Non‐stomatal Limitations Revisited. Ann. Bot. 89 , 183–189 (2002). Baker, N. R. Chlorophyll Fluorescence: A Probe of Photosynthesis In Vivo. Annu. Rev. Plant Biol. 59 , 89–113 (2008). Papadakis, I. E. et al. Mn-induced changes in leaf structure and chloroplast ultrastructure of Citrus volkameriana (L.) plants. J. Plant Physiol. 164 , 100–103 (2007). Zechmann, B. Subcellular Roles of Glutathione in Mediating Plant Defense during Biotic Stress. Plants 9 , 1067 (2020). Thomas, R. Q., Zaehle, S., Templer, P. H. & Goodale, C. L. Global patterns of nitrogen limitation: confronting two global biogeochemical models with observations. Glob. Change Biol. 19 , 2986–2998 (2013). Pottier, M., Masclaux Daubresse, C., Yoshimoto, K. & Thomine, S. Autophagy as a possible mechanism for micronutrient remobilization from leaves to seeds. Front. Plant Sci. 5 , 11 (2014). Kirchhoff, H. Diffusion of molecules and macromolecules in thylakoid membranes. Biochim. Biophys. Acta BBA-Bioenerg. 1837 , 495–502 (2014). Apel, K. & Hirt, H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55 , 373–399 (2004). Eymery, F. & Rey, P. Immunocytolocalization of CDSP 32 and CDSP 34, two chloroplastic drought-induced stress proteins in Solanum tuberosum plants. Plant Physiol. Biochem. 37 , 305–312 (1999). Zhang, M.-P. et al. Changes in chloroplast ultrastructure, fatty acid components of thylakoid membrane and chlorophyll a fluorescence transient in flag leaves of a super-high-yield hybrid rice and its parents during the reproductive stage. J. Plant Physiol. 167 , 277–285 (2010). Fatihi, A. et al. A dedicated type II NADPH dehydrogenase performs the penultimate step in the biosynthesis of vitamin K1 in Synechocystis and Arabidopsis. Plant Cell 27 , 1730–1741 (2015). Spiller, S. & Terry, N. Limiting factors in photosynthesis. II. Iron stress diminishes photochemical capacity by reducing the number of photosynthetic units. Plant Physiol. 65 , 121–5 (1980). Besagni, C. & Kessler, F. A mechanism implicating plastoglobules in thylakoid disassembly during senescence and nitrogen starvation. Planta 237 , 463–470 (2013). Huang, S. et al. Improving nutritional quality of maize proteins by expressing sense and antisense zein genes. J. Agric. Food Chem. 52 , 1958–1964 (2004). Del Rio, L. A., Gomez, M., Yanez, J., Leal, A. & Gorge, J. L. Iron deficiency in pea plants effect on catalase, peroxidase, chlorophyll and proteins of leaves. Plant Soil 49 , 343–353 (1978). Tewari, R. K. Role of mineral nutrient elements in Mulberry (Morus alba L.) Plants with particular reference to oxidative metabolism. (Ph. D thesis, University of Lucknow, INDIA, 2004). Gorbe, E. & Calatayud, Á. Optimization of Nutrition in Soilless Systems: A Review. in Advances in Botanical Research (eds. Kader, J.-C. & Delseny, M.) vol. 53 193–245 (Academic Press, 2010). Valipour, M., Baninasab, B., Khoshgoftarmanesh, A. H. & Gholami, M. Oxidative stress and antioxidant responses to direct and bicarbonate-induced iron deficiency in two quince rootstocks. Sci. Hortic. 261 , 108933 (2020). Hertwig, B., Streb, P. & Feierabend, J. Light dependence of catalase synthesis and degradation in leaves and the influence of interfering stress conditions. Plant Physiol. 100 , 1547–1553 (1992). Wang, J., Zhang, H. & Allen, R. D. Overexpression of an Arabidopsis Peroxisomal Ascorbate Peroxidase Gene in Tobacco Increases Protection Against Oxidative Stress. Plant Cell Physiol. 40 , 725–732 (1999). Shigeoka, S. et al. Regulation and function of ascorbate peroxidase isoenzymes. J. Exp. Bot. 53 , 1305–1319 (2002). Tables Due to technical limitations, all tables are only available as a download in the Supplemental Files section. Supplementary Files Tables.docx Cite Share Download PDF Status: Published Journal Publication published 26 Apr, 2021 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 23 Feb, 2021 Reviews received at journal 03 Feb, 2021 Reviewers agreed at journal 31 Jan, 2021 Reviewers agreed at journal 26 Jan, 2021 Reviewers invited by journal 25 Jan, 2021 Editor assigned by journal 18 Jan, 2021 Editor invited by journal 02 Dec, 2020 Submission checks completed at journal 02 Dec, 2020 First submitted to journal 27 Nov, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-116997","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":5687070,"identity":"28db0e2b-76e2-4354-95df-0000235131c2","order_by":0,"name":"Julie Oustric","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYNACAwYGxvYGBoYEuIgNXvWMDWAtPQeQtaQR0gICEgnIgni08EukP39cUWDHwDzzjdmDh20M8vzSxx8+YEi4h1OL5Iwcw8YzBskMjLNzzA0S2xgMZ/blGBswJBTj1GJwI4exscHgAEiLmUTCmf8JBmd42CQYfyTg0ZL+EKJl5hmQFgagFvbnPxgS8GlJMIRomcED1FIB0sJgxoBPi2TPG8OZDSC/9KSVgbQYzuzhMZZIwKOFnz39wceGP3YMhu2Ht0n+MACGGA/7ww8f8GiBgfqNDchcwhqAQJ4YRaNgFIyCUTAyAQC9t0yX+urjsQAAAABJRU5ErkJggg==","orcid":"","institution":"CNRS, UMR 6134 SPE, Université de Corse","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Oustric","suffix":""},{"id":5687071,"identity":"a08d21c4-a25b-4d21-829d-492933cc94da","order_by":1,"name":"Stéphane Herbette","email":"","orcid":"","institution":"UCA, INRA, PIAF, Clermont-Ferrand, France","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Stéphane","middleName":"","lastName":"Herbette","suffix":""},{"id":5687072,"identity":"d62eed2a-6c4b-4996-8c56-2e54c313e9c1","order_by":2,"name":"Yann Quilichini","email":"","orcid":"","institution":"CNRS, UMR 6134 SPE, Université de Corse","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yann","middleName":"","lastName":"Quilichini","suffix":""},{"id":5687073,"identity":"38a57b71-1fb8-4218-9383-2300b98bda1f","order_by":3,"name":"Raphaël Morillon","email":"","orcid":"","institution":"UMR AGAP, Département BIOS, CIRAD, Station de Roujol","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Raphaël","middleName":"","lastName":"Morillon","suffix":""},{"id":5687074,"identity":"1618a082-b58e-43f6-bd78-2fe4241884da","order_by":4,"name":"Jean Giannettini","email":"","orcid":"","institution":"CNRS, UMR 6134 SPE, Université de Corse","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jean","middleName":"","lastName":"Giannettini","suffix":""},{"id":5687075,"identity":"686b220b-7360-4120-9119-d480668dc009","order_by":5,"name":"Liliane Berti","email":"","orcid":"","institution":"CNRS, UMR 6134 SPE, Université de Corse","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liliane","middleName":"","lastName":"Berti","suffix":""},{"id":5687076,"identity":"7cf1d016-7e76-4d82-b33a-d58504c104e3","order_by":6,"name":"Jérémie Santini","email":"","orcid":"","institution":"CNRS, UMR 6134 SPE, Université de Corse","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jérémie","middleName":"","lastName":"Santini","suffix":""}],"badges":[],"createdAt":"2020-11-27 07:29:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-116997/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-116997/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-021-88383-5","type":"published","date":"2021-04-26T19:03:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":4134587,"identity":"0bfb1fa5-7d72-493d-9c18-343cb1dbcd95","added_by":"auto","created_at":"2020-12-09 19:05:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":747732,"visible":true,"origin":"","legend":"Scanning electron micrographs of abaxial epidermis and stomata in leaves of common clementine scion grafted onto different rootstocks. \nAbaxial epidermis and stomata of leaves of common clementine scion grafted onto diploid (C/CC2x) and doubled diploid (C/CC4x) Carrizo citrange and diploid (C/CM2x) and doubled diploid (C/CM4x) Citrumelo 4475 rootstocks grown in nutrient reference solution (100%) (A, C, E and G, scale bar: 10 µm) and without nutrient solution (0%) (B, D, F and H, scale bar: 10 µm) for 210 days. \nST: ostiole (stoma); GC: guard cells.\n","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-116997/v1/8b66da1fa08a2c9a2549014d.png"},{"id":4134588,"identity":"a8a22bc7-3077-41e6-baf3-b5bf676f2e32","added_by":"auto","created_at":"2020-12-09 19:05:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":680657,"visible":true,"origin":"","legend":"Transmission electron micrographs of palisade mesophyll cells in leaves of common clementine scion grafted onto different rootstocks. \n(A, B, C, D, I, J, K, L; scale bar: 5 µm) Palisade mesophyll cells and (E, F, G, H, M, N, O, P; scale bar: 1 µm) their respective chloroplasts in leaves of common clementine scion grafted onto diploid (C/CC2x) and doubled diploid (C/CC4x) Carrizo citrange and diploid (C/CM2x) and doubled diploid (C/CM4x) Citrumelo 4475 rootstocks grown in nutrient reference solution (100%). St: starch; Pg: plastoglobuli; Mt: mitochondria; Gr: granum.\n","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-116997/v1/929f97031ab28e7ae6538d5d.png"},{"id":4134589,"identity":"82a4baee-5c8a-4f80-b64e-3359066c644d","added_by":"auto","created_at":"2020-12-09 19:05:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":494917,"visible":true,"origin":"","legend":"Transmission electron micrographs of spongy mesophyll cells of leaves of common clementine scion grafted onto different rootstocks. \n(A, B, C, D, I, J, K, L; scale bar: 5 µm) Spongy mesophyll cells and (E, F, G, H, M, N, O, P; scale bar: 1 µm) their respective chloroplasts in leaves of common clementine scion grafted onto diploid (C/CC2x) and doubled diploid (C/CC4x) Carrizo citrange and diploid (C/CM2x) and doubled diploid (C/CM4x) Citrumelo 4475 rootstocks grown in nutrient reference solution (100%).\nSt: starch; Pg: plastoglobuli; Mt: mitochondria; Gr: granum \n","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-116997/v1/b91c360672bad4233c05941b.png"},{"id":4134590,"identity":"bd1d0686-d7a3-4f28-a0d5-7e80bda8cac6","added_by":"auto","created_at":"2020-12-09 19:05:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":117846,"visible":true,"origin":"","legend":"Leaf damages of common clementine scion grafted onto different rootstocks.\nLeaf damages of common clementine scion grafted onto diploid (C/CC2x) and doubled diploid (C/CC4x) Carrizo citrange and diploid (C/CM2x) and doubled diploid (C/CM4x) Citrumelo 4475 rootstocks grown in nutrient reference solution (100%) and without nutrient solution (0%).\n\n","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-116997/v1/66e030b783eb74334916d037.png"},{"id":4134591,"identity":"956919ce-dd7c-4cfb-adb8-1c7a886c20d0","added_by":"auto","created_at":"2020-12-09 19:05:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":36414,"visible":true,"origin":"","legend":"Effect of nutrient deficiency on photosynthetic properties of leaves of common clementine scion grafted onto different rootstocks. \n(A) Leaf net photosynthetic rate (Pnet), (B) stomatal conductance (gs) and (C) chlorophyll fluorescence (Fv/Fm) of leaves of common clementine scion grafted onto diploid (C/CC2x) and doubled diploid (C/CC4x) Carrizo citrange and diploid (C/CM2x) and doubled diploid (C/CM4x) Citrumelo 4475 rootstocks grown in nutrient reference solution (100%) (white circles) and without nutrient solution (0%) (black circles) for 210 days. Values are mean (± standard error) of 9 independent measurements (n = 9) for each scion/rootstock combination, i.e. three per tree. Ploidy and treatment effects were analyzed using ANOVA and Fisher LSD tests (P \u003c 0.05). Distinct letters indicate significant differences between all scion/rootstocks combinations and treatments.\n","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-116997/v1/0e78759a130867d411eb689d.png"},{"id":4134592,"identity":"6dc80e01-cd92-4585-99e9-0a59b744eb11","added_by":"auto","created_at":"2020-12-09 19:05:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":64053,"visible":true,"origin":"","legend":"Effect of nutrient deficiency on antioxidant enzyme activities and contents in oxidative compounds in leaves of common clementine scion grafted onto different rootstocks. \n(A) Superoxide dismutase (SOD), (B) catalase (CAT) and (C) ascorbate peroxidase (APX) specific activities and (D) malondialdehyde (MDA) and (E) H2O2 contents in leaves of common clementine scion grafted onto diploid (C/CC2x) and doubled diploid (C/CC4x) Carrizo citrange and diploid (C/CM2x) and doubled diploid (C/CM4x) Citrumelo 4475 rootstocks grown in nutrient reference solution (100%) (white circles) and without nutrient solution (0%) (black circles) for 210 days. Values are mean (± standard error) of 3 independent measurements from 3 samples for each scion/rootstock combination, i.e. one per tree, obtained by pooling 8 fully-expanded leaves (n = 3). Ploidy and treatment effects were analyzed using ANOVA and Fisher LSD tests (P \u003c 0.05). Distinct letters indicate significant differences between the four scion/rootstocks combinations and treatments.\n\n","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-116997/v1/d948ab49378be3384d23b9f2.png"},{"id":13630221,"identity":"cf4fc060-3871-4dfa-8ee5-12fc065f0ee0","added_by":"auto","created_at":"2021-09-17 08:11:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2729933,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-116997/v1/0aaa93e4-ffbf-4947-a320-75b28c594697.pdf"},{"id":4134586,"identity":"a298e084-2df9-4fba-9388-7cba2d18d9c4","added_by":"auto","created_at":"2020-12-09 19:05:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":62385,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-116997/v1/49b0426d60e3e59a03ac90b4.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eTetraploid Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) Rootstocks Improve Common Clementine Tolerance to Long-term Nutrient Deficiency (Citrus clementina Hort. ex Tan)\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFruit crops, especially citrus fruits, require large amounts of fertilizers to ensure good production and fruit quality. Today, the challenge for sustainable agriculture, and particularly organic agriculture, is to reduce the use of inputs in crops. Reducing inputs optimizes the economic outcome while limiting the environmental impact.\u003c/p\u003e\n\u003cp\u003eWater and minerals absorbed by roots in the soil are essential for plant development, growth and reproduction. Fourteen minerals are considered essential and these can be divided into two groups: the macroelements (N, K, P, Ca, Mg and S) which are constituents of organic matter (proteins, nucleic acids) or play a strong osmotic role, and microelements (Zn, Cu, Fe, Mn, B, Mo, Cl and Ni), which are only involved as specific cofactors or constituents of certain enzymes\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. Mineral-deficient plants present various visual symptoms, such as necrosis, chlorosis, dark green foliage, or stunted growth\u003csup\u003e4\u003c/sup\u003e. Essential mineral deficiency alters plant primary metabolism and this disrupts the physiological and biochemical processes leading to changes in leaf structure and ultrastructure\u003csup\u003e5,6\u003c/sup\u003e. Moreover, cells must cope with an overproduction of reactive oxygen species (ROS) such as singlet oxygen (O\u003csup\u003e*\u003c/sup\u003e), hydroxyl radicals (OH\u003csup\u003e\u0026bull;\u003c/sup\u003e), superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) which cause membrane leakage due to lipid peroxidation and damage to proteins and nucleic acids\u003csup\u003e7\u0026ndash;9\u003c/sup\u003e. As a result, ROS defence mechanisms are activated by a set of antioxidant compounds (metabolites such as ascorbate and proline) and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR)). Adapted genotypes are therefore sought in order to propose cultural itineraries more suitable to low input conditions.\u003c/p\u003e\n\u003cp\u003eIn citrus crops, improving the performance of varieties is based on the scion/rootstock combination forming the aerial parts and roots of the plant, respectively. The impact of the rootstock on the scion lies in its influence on flowering, fruit quality, canopy size, and resistance, among other parameters\u003csup\u003e10\u003c/sup\u003e. Good relations between rootstock and scion are essential for the efficient translocation of water and mineral nutrients and to promote biomass production and tolerance to biotic and abiotic factors, such as nutrient deficiency\u003csup\u003e11,12\u003c/sup\u003e. Improved root system vigour in citrus rootstocks results in increased soil nutrient and water uptake\u003csup\u003e13,14\u003c/sup\u003e. All modern cultivated varieties of citrus are now grafted on diploid rootstocks and therefore have two sets of chromosomes in their genetic heritage. However, incomplete mitosis of somatic embryos may occur in seedlings of diploid (2x) apomictic genotypes with formation of doubled diploid (4x) genotypes\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eRecent advances have shown that rootstock tetraploidy can improve the tolerance of a scion. Grafting of Valencia Delta sweet orange (\u003cem\u003eCitrus sinensis\u003c/em\u003e L.) on 4x rangpur lime (\u003cem\u003eCitrus limonia\u003c/em\u003e) rootstock was found to improve its tolerance to water stress by changing patterns of gene expression in Rangpur lime citrus roots regulating adaptation to water deficit\u003csup\u003e16\u003c/sup\u003e. Natural chilling stress tolerance associated with a robust antioxidant system was also enhanced in common clementine (\u003cem\u003eCitrus clementina\u003c/em\u003e Hort. ex Tan) grafted with Carrizo citrange \u003ca href=\"https://www.sciencedirect.com/science/article/pii/S0176161717301177\"\u003e(\u003cem\u003eCitrus\u003c/em\u003e \u003cem\u003esinensis\u003c/em\u003e Osb.\u0026times; Poncirus trifoliata L. Raf.) 4x rootstock\u003csup\u003e17\u003c/sup\u003e\u003c/a\u003e. Chromium tolerance of Kinnow mandarin (\u003cem\u003eCitrus nobilis\u003c/em\u003e Lour x \u003cem\u003eCitrus deliciosa\u003c/em\u003e Ten) grafted on three 4x rootstocks\u003cem\u003e (Poncirus trifoliata\u003c/em\u003e [L.], \u003cem\u003eCitrus reshni,\u003c/em\u003e and \u003cem\u003eCitrus limonia\u003c/em\u003e Osbeck.) may be attributed to chromium sequestration in roots with lower transfer to leaves in 4x rootstocks\u003csup\u003e18\u003c/sup\u003e. The use of rootstocks better adapted to environmental constraints seems to be a promising eco-friendly strategy.\u003c/p\u003e\n\u003cp\u003eMany \u003cem\u003eCitrus\u003c/em\u003e genotypes are used as rootstock for citrus cultivation. Genotypes belong either to the \u003cem\u003eCitrus\u003c/em\u003e genus such as Volkamer lemon or are obtained by hybridization between \u003cem\u003eCitrus\u003c/em\u003e and \u003cem\u003ePoncirus\u003c/em\u003e genus progenitors such as Citrumelo 4475 and Carrizo citrange. Volkamer lemon which is used as rootstock for lemon, is adapted to dry, calcareous and saline soils and presents tolerance to Tristeza, cachexia and exocortis. Citrumelo 4475 imparts cold tolerance to the scion. Citrange Carrizo is frequently used in acidic and neutral soils but not in dry areas because of its limited performance under drought conditions. These two genotypes inherited Tristeza tolerance from their Trifoliate orange progenitor and give clementine varieties that produce a high yield and fruit quality\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn a previous study, we compared leaf structure and ultrastructure under nutrient deficiency of two genotypes used worldwide as rootstock for citrus cultivation, the Citrumelo 4475 (\u003cem\u003eCitrus paradisi\u003c/em\u003e L. Macf. \u0026times; Poncirus trifoliata L. Raf.) and Volkamer lemon (\u003cem\u003eCitrus limonia\u003c/em\u003e Osb.) with both 2x and 4x genotypes\u003csup\u003e20\u003c/sup\u003e. Results showed an increase in tolerance to nutrient deficiency in 4x genotypes. Doubled diploid genotypes presented less degradation of ultrastructural components such as chloroplasts, thylakoids, mitochondria and starch grains associated with a smaller decrease in leaf net photosynthetic rate (\u003cem\u003eP\u003c/em\u003e\u003csub\u003enet\u003c/sub\u003e), stomatal conductance (\u003cem\u003eg\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e) and chlorophyll fluorescence (\u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) compared to 2x genotypes.\u003c/p\u003e\n\u003cp\u003eThe higher tolerance to nutrient deficiency was linked to the reduced accumulation of malondialdehyde (MDA) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in Citrumelo 4475 4x than in the 2x, implying a more efficient antioxidant system in the 4x genotype. However, few differences in the antioxidant system and oxidative status were observed between 2x and 4x Volkamer lemons.\u003c/p\u003e\n\u003cp\u003eThe aim of this study was therefore to determine the impact of rootstock ploidy level on the 2x common clementine scion during nutrient deficiency. We selected common clementine grafted on two rootstocks used worldwide for clementine cultivation, i.e. Citrumelo 4475 (C/CM2x) and Carrizo citrange (C/CC2x) in both 2x and 4x types (C/CM4x and C/CC4x, respectively). The effect of rootstock ploidy level on common clementine scions under nutrient deficiency was investigated by studying common clementine leaf structure and ultrastructure and physiological and biochemical parameters.\u003c/p\u003e"},{"header":"Results And Discussion ","content":"\u003ch2\u003eEffect of rootstock ploidy level on anatomical properties of common clementines under control conditions\u003c/h2\u003e\n\u003cp\u003eWhatever the rootstock genotype and ploidy level, microscopic examination of leaf surface imprints confirmed the presence of stomata only on their abaxial surface that were surrounded by ordinary epidermal cells\u003csup\u003e29\u003c/sup\u003e (anomocytic organization) (Fig. 1). Rootstock tetraploidy did not induce any changes in the location of stomata or the epidermal cell structure of common clementine leaves. This agrees with this anomocytic organization observed on leaves of tetraploid seedlings\u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eStomata size was unchanged in C/CM4x and C/CC4x combinations. Conversely, stomata density decreased and ostiole sizes increased in C/CC4x compared to C/CC2x combinations (Table 1). Studies have shown a positive correlation between stomata and ostiole size and a negative correlation between stomata density and ploidy level in both 4x ungrafted genotypes and 3x clementine in comparison to their 2x counterparts\u003csup\u003e20,29,30\u003c/sup\u003e. Our study showed that 4x rootstocks had no effect on stomata sizes in 2x common clementine scions and a non-systematic impact on ostiole size and stomatal density, putatively through changes in hydraulics or mineral inputs. The decrease in stomatal density caused by rootstock tetraploidy in the C/CC4x combination was not associated with any changes in gas exchanges compared to its C/CC2x counterpart (Table 1; Fig. 1 and 5). The increase in ostiole size in the C/CC4x combination compared to its C/CC2x counterpart suggests an adjustment of the stomata opening to compensate for the reduced stomatal density and maintain stomatal conductance\u003csup\u003e31\u003c/sup\u003e. This adjustment could be due to the considerable degradation of starch grains in the guard cells in the first hour of light contributing to a rapid increase in the opening of the stomata in parallel with the activation of membrane ion transport\u003csup\u003e32,33\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eDespite identical physical stomatal attributes between C/CM2x and C/CM4x combinations, analysis of the gas exchange parameters revealed a decrease of \u003cem\u003eP\u003c/em\u003e\u003csub\u003enet\u003c/sub\u003e in the C/CM4x combination (Table 1; Fig. 1 and 5). Other factors than leaf structure therefore appear to be involved in the regulation of photosynthesis in the C/CM4x combination.\u003c/p\u003e\n\u003cp\u003eAt ultrastructural level, rootstock tetraploidy initiated changes in the number and size of the main cell components of the 2x common clementine leaves (Tables 2 and 3). Cell size and thickness in both palisade and spongy mesophylls were reduced in C/CC4x compared to C/CC2x, whereas they increased in C/CM4x compared to C/CM2x (Tables 2 and 3; Fig. 2 and 3). These ultrastructural modifications were the only identical changes in C/CM4x and the ungrafted rootstock counterpart\u003csup\u003e20\u003c/sup\u003e. In contrast to its ungrafted counterpart, C/CM4x showed a lower \u003cem\u003eP\u003c/em\u003e\u003csub\u003enet \u003c/sub\u003eand similar chloroplast numbers to C/CM2x\u003csup\u003e20\u003c/sup\u003e (Tables 2 and 3; Fig. 2, 3 and 5A). Chloroplast enlargement in palisade mesophyll brought about by rootstock tetraploidy in common clementine leaves was not associated with an increase in chloroplast numbers. This phenomenon appears to reduce photosynthetic capacity\u003csup\u003e34\u003c/sup\u003e. Overall, under optimal conditions and depending on the rootstock genotypes, the structural and/or ultrastructural modifications of common clementine leaves induced by rootstock tetraploidy could either compensate each other with no change in gas exchanges or induce a modification of gas exchanges. The number and size of transitory starch grains and mitochondria were either reduced or similar between 2x and 4x scion/rootstock combinations in palisade and spongy mesophylls (Tables 2 and 3; Fig. 2 and 3). The number of plastoglobuli increased in C/CM4x and C/CC4x compared to C/CM2x and C/CC2x in the chloroplasts of the palisade and spongy mesophylls. Only the C/CM4x plastoglobuli were larger than those of its 2x counterpart (Tables 2 and 3; Fig. 2 and 3). Thus, rootstock tetraploidy involved a potential increase in lipid (plastoquinone-9 (PQ-9), plastoquinol-9 (PQ-9H2) and a-tocopherol (a-T)) reserves that could not be deposited in the thylakoids of chloroplasts.\u003c/p\u003e\n\u003cp\u003eRootstock tetraploidy can induced modifications in the number and/or size of the investigated ultrastructural components.\u003c/p\u003e\n\u003ch2\u003eDifferences in photosynthetic properties and redox status of scion/rootstock combinations under nutrient stress could be related to their leaf and cell anatomy\u003c/h2\u003e\n\u003cp\u003eComplete starvation resulted in a significant decrease in N but similar or higher levels of P, K, Mg, Ca and Na than controls in all scion/rootstock combinations (Table 4). A concentration effect induced by the transfer of N to other tree areas could explain the increase in P, K and Mg contents in all scion/rootstock combinations\u003csup\u003e35\u003c/sup\u003e. Leaf chlorosis occurs when plants do not have the nutrients needed for chlorophyll synthesis, which in turn affects the photosynthetic efficiency\u003csup\u003e36\u003c/sup\u003e. However, the decrease in N, which was an important factor in chlorosis, was similar in all scion/rootstock combinations (Table 4). C/CM4x showed less chlorosis as indicated by its light green colour compared with the yellow colour of other scion/rootstock combinations (Fig. 4). These results suggest a better integrity of chlorophyll content probably related to a better protection against ROS\u003csup\u003e37\u0026ndash;39\u003c/sup\u003e. The improvements in the redox status of scions by tetraploid rootstocks is probably linked to an improvement in photosynthesis.\u003c/p\u003e\n\u003cp\u003eChlorosis was associated with structural and ultrastructural foliar changes and a disruption of photosynthetic properties in all scion/rootstock combinations\u003csup\u003e6,40\u003c/sup\u003e (Tables 1, 2 and 3; Fig. 5). At structural level, stomata and ostioles of all scion/rootstock combinations showed a narrowing which was associated with an increase in density (except in C/CC2x) response to nutrient deficiency (Table 1; Fig. 1). The rootstock apparently has a different effect on the structural components of common clementine leaves depending on genotype and/or ploidy level. \u003cem\u003eP\u003c/em\u003e\u003csub\u003enet\u003c/sub\u003e and \u003cem\u003eg\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e decreased concomitantly in all scion/rootstock combinations under nutrient deficiency (Fig. 5A-B). These results suggest a critical role of stomatal structure in the process of CO\u003csub\u003e2\u003c/sub\u003e availability\u003csup\u003e41\u003c/sup\u003e. However, the smaller decrease in \u003cem\u003eP\u003c/em\u003e\u003csub\u003enet\u003c/sub\u003e, \u003cem\u003eg\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e and \u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e in C/CM4x than in other scion/rootstock combinations (Fig. 5) implies that other factors than stomata are needed to sustain photosynthesis\u003csup\u003e42\u0026ndash;44\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAt ultrastructural level, nutrient deficiency resulted in cell size enlargement (except in C/CM2x spongy mesophyll), thylakoid with grana degradation and a decrease in chloroplast size in leaf mesophylls of all scion/rootstock combinations (Tables 2 and 3; Fig. 2 and 3). Similar results were recorded in other plants under conditions of high light stress, infection, dark-induced senescence or total nutrient deficiency\u003csup\u003e6,20,45,46\u003c/sup\u003e. Chloroplast degradation may be due to the significant decrease in N in the mature leaves of each scion/rootstock combination following the extensive remobilization of N present in the cell to younger leaves or storage areas during nutrient deficiency\u003csup\u003e47,48\u003c/sup\u003e (Tables 2 and 3; Fig. 2 and 3).\u003c/p\u003e\n\u003cp\u003eThe overproduction of reactive oxygen species (ROS) leads to alterations in the integrity of the membrane structures of cells, chloroplasts or thylakoids by a process called lipid peroxidation, one of the indicators of which is malondialdehyde (MDA). The lower MDA content in C/CM4x compared to other scion/rootstock combinations was consistent with the reduced damage to ultrastructure (Tables 2 and 3; Fig. 2, 3, and 6D). Thylakoid size and structure is dependent on the formation of PSII-LHCII supercomplexes\u003csup\u003e49\u003c/sup\u003e. In C/CM4x, the lower decrease in \u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e suggests a more limited degradation of the PSII-LHCII supercomplexes reducing the disruption of electron transport for the photosynthetic reaction and thus the production of ROS\u003csup\u003e50\u003c/sup\u003e (Fig. 5C and 6D)\u003cem\u003e. \u003c/em\u003eMaintaining the redox balance in C/CM4x decelerates the degradation of thylakoids resulting in a more efficient photosynthetic capacity (\u003cem\u003eP\u003c/em\u003e\u003csub\u003enet\u003c/sub\u003e and \u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) compared to C/CM2x, C/CC2x and C/CC4x after nutrient deficiency (Tables 2 and 3; Fig. 2, 3, 5A, 5C and 6D). Plastoglobuli are bound to thylakoids via their membranes. Studies have shown that plastoglobuli are involved in the formation and degradation of thylakoids during plant growth, development and senescence but also when plants are exposed to drought, high-light, N starvation or fungal infection\u003csup\u003e46,51\u0026ndash;53\u003c/sup\u003e. The reduced damage of the thylakoid membrane in C/CM4x would explain \u003ca href=\"http://www.reverso.net/translationresults.aspx?lang=FR\u0026amp;sourcetext=le%20maintien%20du%20nombre%20et%20la%20faible%20augmentation%20de%20la%20taille%20des%20plastoglobules%20due%20\u0026agrave;%20l\u0026rsquo;accumulation%20des%20lipides%20dans%20leurs%20core%20hydrophobique%20comparativement%20aux%20autres%20scion/rootstock\u0026amp;action_form=translate\u0026amp;direction_translation=fra-eng-7\"\u003ethe maintenance of the plastoglobuli numbers and the small increase in their size \u003c/a\u003edue to the accumulation of catabolites formed by thylakoid degradation in their hydrophobic core compared to other scion/rootstock combinations\u003csup\u003e54,55\u003c/sup\u003e (Tables 2 and 3; Fig. 2 and 3).\u003c/p\u003e\n\u003cp\u003eRootstock tetraploidy improves the tolerance of 2x common clementine scions by maintaining the redox status and delaying ultrastructural changes and damage with a consequent improvement in photosynthetic capacity. However, as suggested by the similar impact of nutrient deficiency on C/CC2x and C/CC4x, rootstock tetraploidy does not automatically result in better tolerance of the photosynthetic properties of the scion.\u003c/p\u003e\n\u003ch2\u003eDifferences in tolerance to nutrient stress between scion/rootstock combinations may be explained by a better antioxidant system\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eNutrient deprivation led to a modification of oxidative metabolism\u003c/em\u003e\u003csup\u003e35,39,56\u003c/sup\u003e\u003cem\u003e. Tolerance differences between scion/rootstock combinations may be related to differences in ROS production and/or antioxidant system efficiency. The enzymatic antioxidant system response differs according to plant species and the deficient minerals\u003c/em\u003e\u003csup\u003e56\u0026ndash;60\u003c/sup\u003e\u003cem\u003e. Enzymatic antioxidant systems in common clementine leaves differed depending on the rootstock genotypes (Fig. 6). \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNutrient deficiency impaired SOD activity in C/CC4x and C/CM4x (Fig. 6A). \u003c/em\u003eIt was interesting to note that APX activity either increased or remained similar to the control in all scion/rootstock combinations (Fig. 6C) whereas CAT activity only increased in C/CM4x under nutrient deficiency \u003cem\u003e(Fig. 6B)\u003c/em\u003e. \u003cem\u003eConcurrent CAT and APX activity is important for the elimination of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/em\u003e\u003csup\u003e61\u0026ndash;63\u003c/sup\u003e\u003cem\u003e. \u003c/em\u003eIn C/CM2x, C/CC2x and C/CC4x, the low H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and high MDA contents were due to increased OH\u003csup\u003e\u0026bull;\u003c/sup\u003e formation (Fig. 6D and E). This OH\u003csup\u003e\u0026bull;\u003c/sup\u003e is either directly formed by the addition of electrons to the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e not transformed into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by SOD and/or by the transformation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e via Fenton or Haber-Weiss reactions when the APX activity is insufficient to compensate for the low CAT activity. \u003cem\u003eConversely, in C/CM4x, the synergistic activity of CAT and APX maintains the MDA content in C/CM4x (Fig. 6B, C \u003c/em\u003eand\u003cem\u003e D).\u003c/em\u003e\u003cem\u003eAs in their ungrafted counterparts, the \u003c/em\u003eincreased enzymatic activity may explain the reduced ultrastructural damage and decrease in photosynthetic activity in C/CM4x in comparison with the other scion/rootstock combinations. The improvement in performance of the enzyme-based antioxidant system in the scion is due to the tetraploidization of the rootstock and the compatibility between rootstock and scion.\u003c/p\u003e\n\u003cp\u003eIn conclusion, rootstock ploidy level had no effect on the structure of the common clementine scion leaves (except stoma density in Carrizo citrange) whereas it induced modifications in the ultrastructural components. The impact of prolonged nutrient deficiency on the structure, ultrastructure, physiology and biochemistry of the common clementine scion differed according to the variety and ploidy level of the rootstock. Among the four scion/rootstock combinations, common clementine grafted with 4x citrumelo 4475 rootstock (C/CM4x) was the most tolerant to nutrient deficiency as indicated by the limited changes in leaf cell structures and photosynthetic activity. The improved tolerance of common clementine grafted with 4x citrumelo 4475 rootstock may be related to a better antioxidant system. Tolerance to nutrient deficiency can therefore be improved by rootstock polyploidy but remains dependent on the rootstock genotype. The next step of this study will be to test the impact of rootstock ploidy level on the quality and yield of clementine fruit under nutrient deficiency.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch2\u003ePlant material and experimental design\u003c/h2\u003e\n\u003cp\u003eThe experiment was carried out on the AREFLEC experimental station located in San Giuliano, Corsica (41\u0026deg; 47' 27\u0026rsquo;\u0026rsquo;N and 09\u0026deg; 23' 40\u0026rsquo;\u0026rsquo;E). 2x common clementine (\u003cem\u003eCitrus clementina\u003c/em\u003e Hort. ex Tan; SRA 92) scion grafted onto one year seedlings of Carrizo citrange (\u003cem\u003eCitrus sinensis \u003c/em\u003eL. Osb\u003cem\u003e. \u0026times; Poncirus trifoliata \u003c/em\u003eL. Raf.) and Citrumelo 4475 (\u003cem\u003eCitrus paradisi \u003c/em\u003eL. Macf.\u003cem\u003e \u0026times; Poncirus trifoliata \u003c/em\u003eL. Raf.) 2x (C/CC2x and C/CM2x, respectively) and their 4x counterparts (C/CC4x and C/CM4x, respectively) were used as source materials. The ploidy status of six seedlings for each combination was first checked by flow 10 cytometry (Partec I, Germany) according to Froelicher et al.\u003csup\u003e21\u003c/sup\u003e. Clonal propagation by nucellar embryogenesis was checked by genotyping using SSR markers as described in Vieira et al.\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe 24 selected scion/rootstock combinations were then grown under identical conditions in vermiculite with fertigation and water (1L/h) for three years in a tunnel greenhouse. The stock solution used for irrigation included: 20-5-10 NPK + 2MgO fertilizer + trace elements according to the recommendations of the French department of agriculture. Seedlings were divided into two blocks: one with reference fertigation (control plants) and the other with irrigation water (without nutrient inputs). A total of three plants of each scion/rootstock combination were randomized by fertigation level (\u003cem\u003en\u003c/em\u003e = 3). The fertigation solutions were prepared and applied with a metering pump. Before starting the experiment, the vermiculite was washed for 48 hours in order to eliminate any nutritional reserves in the pot.\u003c/p\u003e\n\u003cp\u003eAccording to a previous experiment of Oustric et al.\u003csup\u003e20\u003c/sup\u003e, leaf and root samples were collected and physiological measurements made from May to December 2018 at two different times (days): 0 (D0: control plant) and 210 (D210) days after the start of nutritional deprivation. Measurements were made and samples taken from homogeneous plants comprising four branches with fully-expanded leaves developed under stress and control conditions.\u003c/p\u003e\n\u003ch2\u003eScanning electron microscopy (SEM)\u003c/h2\u003e\n\u003cp\u003eScanning electron microscopy measurements were carried out on three leaf pieces per scion/rootstock combination and fertigation level (typically 1 cm\u0026sup2;) (\u003cem\u003en\u003c/em\u003e = 3) cut with a razor blade from mid-laminar areas at between 10:00 and 11:00 am. As described in Oustric et al.\u003csup\u003e20\u003c/sup\u003e, leaves were then immediately fixed in cold (4 \u0026deg;C) 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in a 0.1 M cacodylate buffer at pH 7.2, dehydrated through a graded ethanol series (30%, 50%, 75%, 90% and 100%) and dried under CO\u003csub\u003e2 \u003c/sub\u003ein an Emitech K850 critical point dryer (Quorum Technologies Ltd, Ashford, U.K.). Specimens were mounted on aluminum stubs with carbon double-sided adhesive disks, coated with gold/palladium in a SC7640 sputter coater (Quorum Technologies Ltd, Newhaven, U.K.) and examined under a S-3400N scanning electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) at an accelerating voltage of 5 kV.\u003c/p\u003e\n\u003ch2\u003eTransmission electron microscopy (TEM)\u003c/h2\u003e\n\u003cp\u003eTransmission electron microscopy measurements were carried out on five leaf pieces per scion/rootstock combination and fertigation level (typically 1 mm\u0026sup2;) (\u003cem\u003en\u003c/em\u003e = 5) cut with a razor blade from mid-laminar areas at between 10:00 and 11:00 am. As described in Oustric et al.\u003csup\u003e20\u003c/sup\u003e, leaves were immediately fixed in cold (4 \u0026deg;C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in a 0.1 M cacodylate buffer at pH 7.2, post-fixed in cold (4 \u0026deg;C) 1% osmium tetroxide in the same buffer for 1 h, dehydrated through a graded ethanol series (70% and 100%) and propylene oxide, embedded in Spurr, and polymerized at 60 \u0026deg;C for 24 h. Ultra-thin sections (60\u0026ndash;90 nm) were cut using a Power tome PC ultramicrotome (RMC Boeckeler, Tuscon, U.S.A.). Sections were placed on 200- and 300-mesh copper grids and stained with UranyLess (Delta Micoscopies, France) and lead citrate. They were then examined using a Hitachi H-7650 (Hitachi High-Technologies Corporation, Tokyo, Japan) at an accelerating voltage of 80 kV.\u003c/p\u003e\n\u003cp\u003eMineral content was measured on a pool of eight fully expanded leaves for the three plants per combination and fertigation level (\u003cem\u003en\u003c/em\u003e = 3) between 10:00 and 11:00 am. Fresh leaves were placed in a forced air oven at 65 \u0026plusmn; 10\u0026deg; C overnight and then transferred into a desiccator for cooling. The dehydrated leaves were then sent to the CIRAD \u0026ldquo;Analyses des eaux, sols et v\u0026eacute;g\u0026eacute;taux service unit\u0026rdquo; at Montpellier (France) for analysis of macro- and micro-nutrients.\u003c/p\u003e\n\u003cp\u003eLeaf P, K, Ca, Mg and Na contents were measured using an Agilent 720 simultaneous ICP-OES after double calcination with silica removal by adding hydrofluoric acid.\u003c/p\u003e\n\u003cp\u003eThe leaf total N content was evaluated after combustion using a Leco TruMac N determinator.\u003c/p\u003e\n\u003ch2\u003eMeasurements of gas exchange and chlorophyll a fluorescence\u003c/h2\u003e\n\u003cp\u003eAll measurements were made on three fully developed leaves for each of the three plants per combination and fertigation level (\u003cem\u003en\u003c/em\u003e = 9). A portable photosynthesis system (LI600) was used to measure the leaf net photosynthetic rate (\u003cem\u003eP\u003c/em\u003e\u003csub\u003enet\u003c/sub\u003e), stomatal conductance (\u003cem\u003eg\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e) and transpiration rate (E) at between 7:00 and 11:00 am. The carbon dioxide concentration (CO\u003csub\u003e2\u003c/sub\u003e), airflow rate, light intensity and temperature of the leaf chamber were maintained at 380 \u0026mu;mol.mol\u003csup\u003e-1\u003c/sup\u003e, 500 \u0026mu;mol.s\u003csup\u003e-1\u003c/sup\u003e, 1400 \u0026mu;mol.m\u003csup\u003e-2\u003c/sup\u003e.s\u003csup\u003e-1 \u003c/sup\u003eand 25 \u0026deg;C, respectively.\u003c/p\u003e\n\u003cp\u003eChlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence parameters were measured using an OS1p (Hansatech, Instruments Ltd) at between 9:00 and 11:00 am. Leaves were dark-acclimated for 30 min using special leaf clips. Chlorophyll a fluorescence was recorded after illumination with red actinic light (650nm, 3000 \u0026mu;mol photon.m\u003csup\u003e-2\u003c/sup\u003e.s\u003csup\u003e-1\u003c/sup\u003e) for 1 s and this was used to calculate the maximum fluorescence [\u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = (\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e \u0026ndash; \u003cem\u003eF\u003c/em\u003e\u003csub\u003eo\u003c/sub\u003e)/F\u003csub\u003em\u003c/sub\u003e]\u003csup\u003e23\u003c/sup\u003e. Leaves were exposed to an actinic light to evaluate the current fluorescence yield (\u003cem\u003eF\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e) and the actual light-adapted fluorescence (\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e\u0026prime;). Formulas were applied to this data in order to determine the effective quantum yield of PSII Y(II)\u0026nbsp;=\u0026nbsp;(\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e\u0026rsquo;\u0026minus;\u003cem\u003eF\u003csub\u003es\u003c/sub\u003e\u003c/em\u003e)/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e\u0026rsquo;], the Y(NO) = \u003cem\u003eF\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e], the non-photochemical quenching coefficient [Y(NPQ) = (\u003cem\u003eF\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e\u0026rsquo;)-Y(NO)]\u003csup\u003e24,25\u003c/sup\u003e, and the electron transport rate through PSII [ETR(II) = Y(II) x PAR x 0.5 x 0.84]\u003csup\u003e26\u003c/sup\u003e. The ETR/\u003cem\u003eP\u003c/em\u003e\u003csub\u003enet \u003c/sub\u003eratio was calculated to estimate the use of electrons in other processes unrelated to the photosynthetic CO\u003csub\u003e2\u003c/sub\u003e assimilation rate.\u003c/p\u003e\n\u003ch2\u003eDetermination of oxidative stress and antioxidant levels\u003c/h2\u003e\n\u003cp\u003eBiochemical analyses were performed on three samples for each scion/rootstock combination, i.e. one per tree, obtained by pooling eight fully-expanded leaves (\u003cem\u003en\u003c/em\u003e = 3) collected between 10:00 and 11:00 am and immediately immersed in liquid nitrogen and stored at -80 \u0026deg;C. Immediately prior to biochemical analysis, each leaf and root sample was ground to a fine powder in liquid nitrogen.\u003c/p\u003e\n\u003cp\u003eMalondialdehyde (MDA) and antioxidant enzyme activities (SOD, CAT, and APX) were assayed as defined by Santini et al.\u003csup\u003e27\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eHydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was assayed using the PeroxiDetect kit (Sigma-Aldrich). This technique is based on the oxidation of ferrous (Fe\u003csup\u003e2+\u003c/sup\u003e) to ferric ions (Fe\u003csup\u003e3+\u003c/sup\u003e) by hydroperoxides which react with xylenol orange (\u0026ldquo;3,3\u0026prime;-bis[N,N-bis(carboxymethyl)aminomethyl] o-cresolsulfonephthalein, sodium salt\u0026rdquo;) to form a blue complex visible at 560 nm.\u003c/p\u003e\n\u003cp\u003eProline content was measured as described by Oustric et al.\u003csup\u003e28\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eA V-630 spectrophotometer was used for all measurements (Jasco Inc., Tokyo, Japan).\u003c/p\u003e\n\u003ch2\u003eStatistical analyses\u003c/h2\u003e\n\u003cp\u003eAll statistical measurements were performed with R statistical software (v.2.12.1) (\u003ca href=\"http://www.R-project.org\"\u003ehttp://www.R-project.org\u003c/a\u003e) and the Rcmdr package. The qualitative factors studied were sampling date (D0 and D210 after nutrient deficiency), the comment clementine scion grafted onto rootstocks subjected to nutrient stress (C/CC and C/CM) and the ploidy level of nutrient stressed rootstocks (C/CC2x, C/CC4x, C/CM2x and C/CM4x). The influence of these three factors was analyzed using a two-way ANOVA followed by LSD test at \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003eThe microscopic data obtained on leaves of common clementine scion grafted onto the various rootstocks at D0 (control) and D210 of nutrient deficiency were analyzed by heatmaps generated by Heatmap.2 function of the gplot package 3.0.1 for Rstudio (v.1.3.1093) (https://rstudio.com).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis work was funded by the \u0026ldquo;Collectivit\u0026eacute; de Corse\u0026rdquo; as part of the \u0026ldquo;Innov\u0026rsquo;agrumes\u0026rdquo; research project (FEDER). We thank Jean-Claude Ribaut for hosting this study at the AREFLEC experimental station, and Gilles Paolacci and Paul Martin for installing and monitoring the fertigation system in the greenhouse.\u003c/p\u003e\n\u003ch2\u003eAuthor information\u003c/h2\u003e\n\u003cp\u003eAffiliations\u003c/p\u003e\n\u003cp\u003eLaboratoire Biochimie et Biologie Mol\u0026eacute;culaire du V\u0026eacute;g\u0026eacute;tal, CNRS, UMR 6134 SPE, Universit\u0026eacute; de Corse, Corte, France\u003c/p\u003e\n\u003cp\u003eJulie Oustric, Jean Giannettini, Liliane Berti \u0026amp; J\u0026eacute;r\u0026eacute;mie Santini\u003c/p\u003e\n\u003cp\u003eUCA, INRA, PIAF, Clermont-Ferrand, France\u003c/p\u003e\n\u003cp\u003eSt\u0026eacute;phane Herbette\u003c/p\u003e\n\u003cp\u003eLaboratoire Parasites et Ecosyst\u0026egrave;mes M\u0026eacute;diterran\u0026eacute;ens\", CNRS, UMR 6134 SPE, Universit\u0026eacute; de Corse, Corte, France\u003c/p\u003e\n\u003cp\u003eYann Quilichini\u003c/p\u003e\n\u003cp\u003eEquipe \"Am\u0026eacute;lioration des Plantes \u0026agrave; Multiplication V\u0026eacute;g\u0026eacute;tative\", UMR AGAP, D\u0026eacute;partement BIOS, CIRAD, Station de Roujol, Petit-Bourg, Guadeloupe\u003c/p\u003e\n\u003cp\u003eRaphael Morillon\u003c/p\u003e\n\u003ch2\u003eContributions\u003c/h2\u003e\n\u003cp\u003eO.J. collected data, did the statistical analysis, interpreted the results and drafted the manuscript.\u003c/p\u003e\n\u003cp\u003eS.J. designed the study and drafted the manuscript.\u003c/p\u003e\n\u003cp\u003eQ.Y. participated collect microscopic data.\u003c/p\u003e\n\u003cp\u003eM.R., H.S., G.J. and B.L. designed the study and helped draft the manuscript.\u003c/p\u003e\n\u003ch2\u003eEthics declarations\u003c/h2\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMarschner, H. Mineral nutrition of higher plants 2nd edition. \u003cem\u003eAcad. G. B.\u003c/em\u003e (1995).\u003c/li\u003e\n\u003cli\u003eGrusak, M. A. Plant Macro-and Micronutrient Minerals. \u003cem\u003eE LS\u003c/em\u003e (2001).\u003c/li\u003e\n\u003cli\u003eLal, R. Enhancing crop yields in the developing countries through restoration of the soil organic carbon pool in agricultural lands. \u003cem\u003eLand Degrad. Dev.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 197\u0026ndash;209 (2006).\u003c/li\u003e\n\u003cli\u003eEricsson, T. Growth and shoot: root ratio of seedlings in relation to nutrient availability. in \u003cem\u003eNutrient uptake and cycling in forest ecosystems\u003c/em\u003e 205\u0026ndash;214 (Springer, 1995).\u003c/li\u003e\n\u003cli\u003eKalaji, H. M. \u003cem\u003eet al.\u003c/em\u003e Identification of nutrient deficiency in maize and tomato plants by in vivo chlorophyll a fluorescence measurements. \u003cem\u003ePlant Physiol. Biochem.\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 16\u0026ndash;25 (2014).\u003c/li\u003e\n\u003cli\u003eDu, Q. \u003cem\u003eet al.\u003c/em\u003e Effects of potassium deficiency on photosynthesis, chloroplast ultrastructure, ROS, and antioxidant activities in maize (\u003cem\u003eZea mays \u003c/em\u003eL.). \u003cem\u003eJ. Integr. Agric.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 395\u0026ndash;406 (2019).\u003c/li\u003e\n\u003cli\u003eMittler, R. Oxidative stress, antioxidants and stress tolerance. \u003cem\u003eTrends Plant Sci.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 405\u0026ndash;410 (2002).\u003c/li\u003e\n\u003cli\u003eMittler, R., Vanderauwera, S., Gollery, M. \u0026amp; Van Breusegem, F. Reactive oxygen gene network of plants. \u003cem\u003eTrends Plant Sci.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 490\u0026ndash;498 (2004).\u003c/li\u003e\n\u003cli\u003eApel, K. \u0026amp; Hirt, H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. \u003cem\u003eAnnu. Rev. Plant Biol.\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 373\u0026ndash;399 (2004).\u003c/li\u003e\n\u003cli\u003eKucukyumuk, Z. \u0026amp; Erdal, I. Rootstock and cultivar effect on mineral nutrition, seasonal nutrient variation and correlations among leaf, flower and fruit nutrient concentrations in apple trees. \u003cem\u003eBulg. J. Agric. Sci.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 633\u0026ndash;641 (2011).\u003c/li\u003e\n\u003cli\u003eMart\u0026iacute;nez-Ballesta, M. C., Alcaraz-L\u0026oacute;pez, C., Muries, B., Mota-Cadenas, C. \u0026amp; Carvajal, M. Physiological aspects of rootstock\u0026ndash;scion interactions. \u003cem\u003eSci. Hortic.\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 112\u0026ndash;118 (2010).\u003c/li\u003e\n\u003cli\u003eSchwarz, D., Rouphael, Y., Colla, G. \u0026amp; Venema, J. H. Grafting as a tool to improve tolerance of vegetables to abiotic stresses: Thermal stress, water stress and organic pollutants. \u003cem\u003eSci. Hortic.\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 162\u0026ndash;171 (2010).\u003c/li\u003e\n\u003cli\u003eTaylor, B. K. \u0026amp; Dimsey, R. T. Rootstock and scion effects on the leaf nutrient composition of citrus trees. \u003cem\u003eAust. J. Exp. Agric.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 363\u0026ndash;371 (1993).\u003c/li\u003e\n\u003cli\u003eLu, Z.-J. \u003cem\u003eet al.\u003c/em\u003e The effects of inarching \u003cem\u003eCitrus reticulata \u003c/em\u003eBlanco var. tangerine on the tree vigor, nutrient status and fruit quality of \u003cem\u003eCitrus sinensis\u003c/em\u003e Osbeck \u0026lsquo;Newhall\u0026rsquo;trees that have Poncirus trifoliata (L.) Raf. as rootstocks. \u003cem\u003eSci. Hortic.\u003c/em\u003e \u003cstrong\u003e256\u003c/strong\u003e, 108600 (2019).\u003c/li\u003e\n\u003cli\u003eCameron, J. Genetics, breeding and nucellar embryony. \u003cem\u003eCitrus Ind.\u003c/em\u003e 325\u0026ndash;370 (1968).\u003c/li\u003e\n\u003cli\u003eAllario, T. \u003cem\u003eet al.\u003c/em\u003e Tetraploid Rangpur lime rootstock increases drought tolerance via enhanced constitutive root abscisic acid production. vol. 36 (2013).\u003c/li\u003e\n\u003cli\u003eOustric, J. \u003cem\u003eet al.\u003c/em\u003e Tetraploid Carrizo citrange rootstock (\u003cem\u003eCitrus sinensis\u003c/em\u003e Osb. \u0026times; Poncirus trifoliata L. Raf.) enhances natural chilling stress tolerance of common clementine (\u003cem\u003eCitrus clementina\u003c/em\u003e Hort. \u003cem\u003eEx Tan J. Plant Physiol.\u003c/em\u003e \u003cstrong\u003e214\u003c/strong\u003e, 108\u0026ndash;115 (2017).\u003c/li\u003e\n\u003cli\u003eBalal, R. M. \u003cem\u003eet al.\u003c/em\u003e Kinnow mandarin plants grafted on tetraploid rootstocks are more tolerant to Cr-toxicity than those grafted on its diploids one. \u003cem\u003eEnviron. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e140\u003c/strong\u003e, 8\u0026ndash;18 (2017).\u003c/li\u003e\n\u003cli\u003eJacquemond, C., Curk, F. \u0026amp; Heuzet, M. \u003cem\u003eLes cl\u0026eacute;mentiniers et autres petits agrumes\u003c/em\u003e. (Editions Quae, 2013).\u003c/li\u003e\n\u003cli\u003eOustric, J. \u003cem\u003eet al.\u003c/em\u003e Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. \u003cem\u003ePlant Physiol. Biochem.\u003c/em\u003e \u003cstrong\u003e135\u003c/strong\u003e, 372\u0026ndash;384 (2019).\u003c/li\u003e\n\u003cli\u003eFroelicher, Y. \u003cem\u003eet al.\u003c/em\u003e Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets. \u003cem\u003ePlant Cell Rep.\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 937\u0026ndash;944 (2007).\u003c/li\u003e\n\u003cli\u003eVieira, D. D. S. S. \u003cem\u003eet al.\u003c/em\u003e Polyploidization alters constitutive content of volatile organic compounds (VOC) and improves membrane stability under water deficit in Volkamer lemon (\u003cem\u003eCitrus limonia\u003c/em\u003e Osb.) leaves. \u003cem\u003eEnviron. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e, 1\u0026ndash;9 (2016).\u003c/li\u003e\n\u003cli\u003eMaxwell, K. \u0026amp; Johnson, G. N. Chlorophyll fluorescence\u0026mdash;a practical guide. \u003cem\u003eJ. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 659\u0026ndash;668 (2000).\u003c/li\u003e\n\u003cli\u003eKramer, D. M., Johnson, G., Kiirats, O. \u0026amp; Edwards, G. E. New fluorescence parameters for the determination of Q A redox state and excitation energy fluxes. \u003cem\u003ePhotosynth. Res.\u003c/em\u003e \u003cstrong\u003e79\u003c/strong\u003e, 209 (2004).\u003c/li\u003e\n\u003cli\u003eKlughammer, C. \u0026amp; Schreiber, U. Complementary PS II quantum yields calculated from simple fluorescence parameters measured by PAM fluorometry and the Saturation Pulse method. \u003cem\u003ePAM Appl. Notes\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 201\u0026ndash;247 (2008).\u003c/li\u003e\n\u003cli\u003eBilger, W., Schreiber, U. \u0026amp; Bock, M. Determination of the quantum efficiency of photosystem II and of non-photochemical quenching of chlorophyll fluorescence in the field. \u003cem\u003eOecologia\u003c/em\u003e \u003cstrong\u003e102\u003c/strong\u003e, 425\u0026ndash;432 (1995).\u003c/li\u003e\n\u003cli\u003eSantini, J. \u003cem\u003eet al.\u003c/em\u003e Comparison of photosynthesis and antioxidant performance of several \u003cem\u003eCitrus\u003c/em\u003e and \u003cem\u003eFortunella\u003c/em\u003e species (Rutaceae) under natural chilling stress. \u003cem\u003eTrees\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 71\u0026ndash;83 (2013).\u003c/li\u003e\n\u003cli\u003eOustric, J. \u003cem\u003eet al.\u003c/em\u003e Nutrient deficiency tolerance in citrus is dependent on genotype or ploidy level. \u003cem\u003eFront. Plant Sci.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 127 (2019).\u003c/li\u003e\n\u003cli\u003ePadoan, D., Mossad, A., Chiancone, B., Germana, M. A. \u0026amp; Khan, P. S. S. V. Ploidy levels in Citrus clementine affects leaf morphology, stomatal density and water content. \u003cem\u003eTheor. Exp. Plant Physiol.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 283\u0026ndash;290 (2013).\u003c/li\u003e\n\u003cli\u003eJaskani, M. J., Khan, M. M. \u0026amp; Khan, I. A. Growth, morphology and fruit comparison of diploid and tetraploid Kinnow mandarin. \u003cem\u003ePak. J. Agric. Sci.\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 126\u0026ndash;128 (2002).\u003c/li\u003e\n\u003cli\u003eB\u0026uuml;ssis, D., von Groll, U., Fisahn, J. \u0026amp; Altmann, T. Stomatal aperture can compensate altered stomatal density in Arabidopsis thaliana at growth light conditions. \u003cem\u003eFunct. Plant Biol.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 1037\u0026ndash;1043 (2006).\u003c/li\u003e\n\u003cli\u003eSack, F. D. \u0026amp; Paolillo Jr, D. J. Protoplasmic changes during stomatal development in Funaria. \u003cem\u003eCan. J. Bot.\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 2515\u0026ndash;2526 (1983).\u003c/li\u003e\n\u003cli\u003eHorrer, D. \u003cem\u003eet al.\u003c/em\u003e Blue light induces a distinct starch degradation pathway in guard cells for stomatal opening. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 362\u0026ndash;370 (2016).\u003c/li\u003e\n\u003cli\u003eOkie, J. G., Smith, V. H. \u0026amp; Martin-Cereceda, M. Major evolutionary transitions of life, metabolic scaling and the number and size of mitochondria and chloroplasts. \u003cem\u003eProc. R. Soc. B Biol. Sci.\u003c/em\u003e \u003cstrong\u003e283\u003c/strong\u003e, 20160611 (2016).\u003c/li\u003e\n\u003cli\u003eTewari, R. K., Kumar, P. \u0026amp; Sharma, P. N. Oxidative stress and antioxidant responses in young leaves of mulberry plants grown under nitrogen, phosphorus or potassium deficiency. \u003cem\u003eJ. Integr. Plant Biol.\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 313\u0026ndash;322 (2007).\u003c/li\u003e\n\u003cli\u003eS\u0026aacute;rv\u0026aacute;ri, \u0026Eacute;. Effect of Cd on the iron re-supply-induced formation of chlorophyll-protein complexes in cucumber. \u003cem\u003eActa Biol. Szeged.\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 183\u0026ndash;186 (2008).\u003c/li\u003e\n\u003cli\u003eShimshi, D. Leaf chlorosis and stomatal aperture. \u003cem\u003eNew Phytol.\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 455\u0026ndash;461 (1967).\u003c/li\u003e\n\u003cli\u003eRodriguez de Cianzio, S., Fehr, de W. \u0026amp; Anderson, I. C. Genotypic Evaluation for Iron Deficiency Chlorosis in Soybeans by Visual Scores and Chlorophyll Concentration 1. \u003cem\u003eCrop Sci.\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 644\u0026ndash;646 (1979).\u003c/li\u003e\n\u003cli\u003eCakmak, S., G\u0026uuml;l\u0026uuml;t, K. Y., Marschner, H. \u0026amp; Graham, R. D. Effect of zinc and iron deficiency on phytos1derophore release in wheat genotypes differing in zinc efficiency. \u003cem\u003eJ. Plant Nutr.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1\u0026ndash;17 (1994).\u003c/li\u003e\n\u003cli\u003eWu, X., Lu, X., Riaz, M., Yan, L. \u0026amp; Jiang, C. Boron toxicity induced specific changes of cell ultrastructure and architecture of components in leaf center and tip of trifoliate orange [Poncirus trifoliata (L.) Raf.]. \u003cem\u003eJ. Environ. Manage.\u003c/em\u003e \u003cstrong\u003e246\u003c/strong\u003e, 426\u0026ndash;433 (2019).\u003c/li\u003e\n\u003cli\u003eFlexas, J. \u003cem\u003eet al.\u003c/em\u003e Diffusional conductances to CO2 as a target for increasing photosynthesis and photosynthetic water-use efficiency. \u003cem\u003ePhotosynth. Res.\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 45\u0026ndash;59 (2013).\u003c/li\u003e\n\u003cli\u003eEpron, D. \u0026amp; Dreyer, E. Stomatal and non stomatal limitation of photosynthesis by leaf water deficits in three oak species: a comparison of gas exchange and chlorophyll a fluorescence data. \u003cem\u003eAnn. Sci. For.\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 435\u0026ndash;450 (1990).\u003c/li\u003e\n\u003cli\u003eFlexas, J. \u0026amp; Medrano, H. Drought‐inhibition of Photosynthesis in C3 Plants: Stomatal and Non‐stomatal Limitations Revisited. \u003cem\u003eAnn. Bot.\u003c/em\u003e \u003cstrong\u003e89\u003c/strong\u003e, 183\u0026ndash;189 (2002).\u003c/li\u003e\n\u003cli\u003eBaker, N. R. Chlorophyll Fluorescence: A Probe of Photosynthesis In Vivo. \u003cem\u003eAnnu. Rev. Plant Biol.\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 89\u0026ndash;113 (2008).\u003c/li\u003e\n\u003cli\u003ePapadakis, I. E. \u003cem\u003eet al.\u003c/em\u003e Mn-induced changes in leaf structure and chloroplast ultrastructure of \u003cem\u003eCitrus volkameriana\u003c/em\u003e (L.) plants. \u003cem\u003eJ. Plant Physiol.\u003c/em\u003e \u003cstrong\u003e164\u003c/strong\u003e, 100\u0026ndash;103 (2007).\u003c/li\u003e\n\u003cli\u003eZechmann, B. Subcellular Roles of Glutathione in Mediating Plant Defense during Biotic Stress. \u003cem\u003ePlants\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1067 (2020).\u003c/li\u003e\n\u003cli\u003eThomas, R. Q., Zaehle, S., Templer, P. H. \u0026amp; Goodale, C. L. Global patterns of nitrogen limitation: confronting two global biogeochemical models with observations. \u003cem\u003eGlob. Change Biol.\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 2986\u0026ndash;2998 (2013).\u003c/li\u003e\n\u003cli\u003ePottier, M., Masclaux Daubresse, C., Yoshimoto, K. \u0026amp; Thomine, S. Autophagy as a possible mechanism for micronutrient remobilization from leaves to seeds. \u003cem\u003eFront. Plant Sci.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 11 (2014).\u003c/li\u003e\n\u003cli\u003eKirchhoff, H. Diffusion of molecules and macromolecules in thylakoid membranes. \u003cem\u003eBiochim. Biophys. Acta BBA-Bioenerg.\u003c/em\u003e \u003cstrong\u003e1837\u003c/strong\u003e, 495\u0026ndash;502 (2014).\u003c/li\u003e\n\u003cli\u003eApel, K. \u0026amp; Hirt, H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. \u003cem\u003eAnnu. Rev. Plant Biol.\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 373\u0026ndash;399 (2004).\u003c/li\u003e\n\u003cli\u003eEymery, F. \u0026amp; Rey, P. Immunocytolocalization of CDSP 32 and CDSP 34, two chloroplastic drought-induced stress proteins in Solanum tuberosum plants. \u003cem\u003ePlant Physiol. Biochem.\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 305\u0026ndash;312 (1999).\u003c/li\u003e\n\u003cli\u003eZhang, M.-P. \u003cem\u003eet al.\u003c/em\u003e Changes in chloroplast ultrastructure, fatty acid components of thylakoid membrane and chlorophyll a fluorescence transient in flag leaves of a super-high-yield hybrid rice and its parents during the reproductive stage. \u003cem\u003eJ. Plant Physiol.\u003c/em\u003e \u003cstrong\u003e167\u003c/strong\u003e, 277\u0026ndash;285 (2010).\u003c/li\u003e\n\u003cli\u003eFatihi, A. \u003cem\u003eet al.\u003c/em\u003e A dedicated type II NADPH dehydrogenase performs the penultimate step in the biosynthesis of vitamin K1 in Synechocystis and Arabidopsis. \u003cem\u003ePlant Cell\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1730\u0026ndash;1741 (2015).\u003c/li\u003e\n\u003cli\u003eSpiller, S. \u0026amp; Terry, N. Limiting factors in photosynthesis. II. Iron stress diminishes photochemical capacity by reducing the number of photosynthetic units. \u003cem\u003ePlant Physiol.\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 121\u0026ndash;5 (1980).\u003c/li\u003e\n\u003cli\u003eBesagni, C. \u0026amp; Kessler, F. A mechanism implicating plastoglobules in thylakoid disassembly during senescence and nitrogen starvation. \u003cem\u003ePlanta\u003c/em\u003e \u003cstrong\u003e237\u003c/strong\u003e, 463\u0026ndash;470 (2013).\u003c/li\u003e\n\u003cli\u003eHuang, S. \u003cem\u003eet al.\u003c/em\u003e Improving nutritional quality of maize proteins by expressing sense and antisense zein genes. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 1958\u0026ndash;1964 (2004).\u003c/li\u003e\n\u003cli\u003eDel Rio, L. A., Gomez, M., Yanez, J., Leal, A. \u0026amp; Gorge, J. L. Iron deficiency in pea plants effect on catalase, peroxidase, chlorophyll and proteins of leaves. \u003cem\u003ePlant Soil\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 343\u0026ndash;353 (1978).\u003c/li\u003e\n\u003cli\u003eTewari, R. K. Role of mineral nutrient elements in Mulberry (Morus alba L.) Plants with particular reference to oxidative metabolism. (Ph. D thesis, University of Lucknow, INDIA, 2004).\u003c/li\u003e\n\u003cli\u003eGorbe, E. \u0026amp; Calatayud, \u0026Aacute;. Optimization of Nutrition in Soilless Systems: A Review. in \u003cem\u003eAdvances in Botanical Research\u003c/em\u003e (eds. Kader, J.-C. \u0026amp; Delseny, M.) vol. 53 193\u0026ndash;245 (Academic Press, 2010).\u003c/li\u003e\n\u003cli\u003eValipour, M., Baninasab, B., Khoshgoftarmanesh, A. H. \u0026amp; Gholami, M. Oxidative stress and antioxidant responses to direct and bicarbonate-induced iron deficiency in two quince rootstocks. \u003cem\u003eSci. Hortic.\u003c/em\u003e \u003cstrong\u003e261\u003c/strong\u003e, 108933 (2020).\u003c/li\u003e\n\u003cli\u003eHertwig, B., Streb, P. \u0026amp; Feierabend, J. Light dependence of catalase synthesis and degradation in leaves and the influence of interfering stress conditions. \u003cem\u003ePlant Physiol.\u003c/em\u003e \u003cstrong\u003e100\u003c/strong\u003e, 1547\u0026ndash;1553 (1992).\u003c/li\u003e\n\u003cli\u003eWang, J., Zhang, H. \u0026amp; Allen, R. D. Overexpression of an Arabidopsis Peroxisomal Ascorbate Peroxidase Gene in Tobacco Increases Protection Against Oxidative Stress. \u003cem\u003ePlant Cell Physiol.\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 725\u0026ndash;732 (1999).\u003c/li\u003e\n\u003cli\u003eShigeoka, S. \u003cem\u003eet al.\u003c/em\u003e Regulation and function of ascorbate peroxidase isoenzymes. \u003cem\u003eJ. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 1305\u0026ndash;1319 (2002).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eDue to technical limitations, all tables are only available as a download in the Supplemental Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Nutrient deficiency, nutritious food, Citrus crops, genotype","lastPublishedDoi":"10.21203/rs.3.rs-116997/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-116997/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Nutrient deficiency alters growth and the production of high-quality nutritious food. In Citrus crops, rootstock technologies have become a key tool for enhancing tolerance to abiotic stress. The use of doubled diploid rootstocks can improve adaptation to lower nutrient inputs. This study investigated leaf structure and ultrastructure and physiological and biochemical parameters of common clementine scions (C) grafted on diploid (2x) and doubled diploid (4x) Carrizo citrange (C/CC2x and C/CC4x) and Citrumelo 4475 (C/CM2x and C/CM4x) rootstocks under optimal fertigation and after seven months of nutrient deficiency. Rootstock ploidy level had no impact on structure but induced changes in the number and/or size of cells and some cell components of common clementine leaves under optimal nutrition. Rootstock ploidy level did not modify gas exchanges in Carrizo citrange but induced a reduction in the leaf net photosynthetic rate in Citrumelo 4475. By assessing foliar damage, changes in photosynthetic processes and malondialdehyde accumulation, we found that C/CM4x were less affected by nutrient deficiency than the other scion/rootstock combinations. Their greater tolerance to nutrient deficiency was probably due to the better performance of the enzyme-based antioxidant system. Nutrient deficiency had similar impacts on C/CC2x and C/CC4x. Tolerance to nutrient deficiency can therefore be improved by rootstock polyploidy but remains dependent on the rootstock genotype.\n","manuscriptTitle":"Tetraploid Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) Rootstocks Improve Common Clementine Tolerance to Long-term Nutrient Deficiency (Citrus clementina Hort. ex Tan)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-09 19:05:08","doi":"10.21203/rs.3.rs-116997/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-02-23T09:14:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-02-03T10:40:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4738bd31-f90c-4cb4-ab1c-d4366c33376e","date":"2021-01-31T17:29:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7e3542ac-530c-4f38-8216-1bde6ac082dd","date":"2021-01-26T10:13:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-01-25T14:21:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-01-18T13:40:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-12-02T09:30:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-12-02T07:19:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2020-11-27T07:19:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ab88b928-abf5-402a-ba6f-4d473de077e2","owner":[],"postedDate":"December 9th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1404017,"name":"Space Exploration"},{"id":1404018,"name":"Mechanical Engineering"}],"tags":[],"updatedAt":"2021-08-18T19:15:59+00:00","versionOfRecord":{"articleIdentity":"rs-116997","link":"https://doi.org/10.1038/s41598-021-88383-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2021-04-26 19:03:05","publishedOnDateReadable":"April 26th, 2021"},"versionCreatedAt":"2020-12-09 19:05:08","video":"","vorDoi":"10.1038/s41598-021-88383-5","vorDoiUrl":"https://doi.org/10.1038/s41598-021-88383-5","workflowStages":[]},"version":"v1","identity":"rs-116997","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-116997","identity":"rs-116997","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}