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 CO2 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 (Pnet), stomatal conductance (gs) and transpiration rate (E) at between 7:00 and 11:00 am. The carbon dioxide concentration (CO2), 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 [Fv/Fm = (Fm – Fo)/Fm]23. Leaves were exposed to an actinic light to evaluate the current fluorescence yield (Fs) and the actual light-adapted fluorescence (Fm′). Formulas were applied to this data in order to determine the effective quantum yield of PSII Y(II) = (Fm’−Fs)/Fm’], the Y(NO) = Fs/Fm], the non-photochemical quenching coefficient [Y(NPQ) = (Fs/Fm’)-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/Pnet ratio was calculated to estimate the use of electrons in other processes unrelated to the photosynthetic CO2 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 (H2O2) was assayed using the PeroxiDetect kit (Sigma-Aldrich). This technique is based on the oxidation of ferrous (Fe2+) to ferric ions (Fe3+) 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).