DOI: 10.5061/dryad.x3ffbg7nj
title: Data from: Nitrogen availability and plant-plant interactions drive leaf silicon concentration in wheat genotypes

description: Estimating plasticity of leaf silicon (Si) in response to abiotic and
 biotic factors underpins our comprehension of plant defences and stress
 resistance in natural and agroecosystems. However, how nitrogen (N)
 addition and intraspecific plant-plant interactions affect Si accumulation
 remains unclear. We grew 19 durum wheat genotypes (Triticum turgidum ssp.
 durum) in pots, either alone, or in intra- or intergenotypic cultures of
 two individuals, and with or without N. Aboveground biomass, plant height
 and leaf [Si] were quantified at the beginning of the flowering stage.
 Nitrogen addition decreased leaf [Si] for most genotypes, proportionally
 to the biomass increase. Si plasticity to plant-plant interactions varied
 significantly among genotypes, with both increases and decreases in leaf
 [Si] when mixed with a neighbour, regardless of the mixture type
 (intra-/intergenotype). Besides, increased leaf [Si] in response to
 plant-plant interactions was associated with increased plant height. Our
 results suggest the occurrence of both facilitation and competition for Si
 uptake from the rhizosphere in wheat mixtures. Future research should
 identify which leaf and root traits characterize facilitating neighbours
 for Si acquisition. We also show that Si could be involved in height gain
 in response to intraspecific competition, possibly for increasing light
 capture. This important finding opens up new research directions on Si and
 plant-plant interactions in both natural ecosystems and agroecosystems.
 More generally, our results stress the need to explore leaf Si plasticity
 in responses to both abiotic and biotic factors to understand plant stress
 resistance.
description: <em>Experimental
 design</em> We selected 19 durum wheat genotypes
 [<em>Triticum
 turgidum</em> ssp. <em>Durum </em>(Desf.)] from the Evolutionary Pre-breeding
 pOpulation (EPO), a population of 180 genotypes with high phenotypic and
 genotypic diversities (David et al.,
 2014). The 19 genotypes represented a large
 phenotypic diversity on below- and above-ground traits. The 19 genotypes
 were grown either alone in <em>single</em> (alone in the pot), in
 <em>intragenotypic
 culture</em> (two plants of the same genotype in the
 same pot), or in <em>intergenotypic
 culture</em> (two plants from different genotypes in the
 same pot), hereafter <em>growth
 modalities</em>, with two levels of N (treatment
 N<sup>+</sup> and N<sup>-</sup>), and in
 triplicate. We randomly
 assembled 26 <em>intergenotypic
 </em>mixtures among the 171 possibilities.
 The modality <em>single</em> thus represents 114 individuals (19
 genotypes * 2 N levels * 3 replicates), <em>intragenotypic culture
 </em>228 individuals (2 plants * 19 genotypes * 2
 N levels * 3 replicates), and <em>intergenotypic
 culture</em> 312 individuals (26 mixtures of 2 plants *
 2 N levels * 3 replicates). In total, 384 pots and 654
 wheat individuals were considered. <em>Growth
 conditions</em> The experiment was conducted at the CEFE
 experimental field (Montpellier, France) from January to May 2021, in
 outdoor conditions. We used a randomised complete block design using three
 blocks (one replicate in each block). Plants were grown in 4-L
 plastic pots (18.5 cm diameter; 21.5 cm depth) filled with approximately
 4.5 kilos of local soil (52% sand, 27% silt and 21%
 clay; 6.9% CaCO<sub>3</sub>; 4.1% organic carbon; 0.21% total
 N; pH 8.0), and amended with PK fertiliser (0.38 g
 pot<sup>-1</sup>;
 P<sub>2</sub>O<sub>5</sub> and
 K<sub>2</sub>O). The effect of
 plant-plant interactions on plant Si uptake might be influenced by soil Si
 availability (Ning et al.,
 2021). Here, although not quantified, we expect Si availability to be rather high in this young, high-pH and clay+silt-rich soil (Cornelis & Delvaux, 2016). Indeed, a recent analysis of soil Si availability in French soils shows that this soil type exhibits the highest Si concentrations extracted with CaCl<sub>2</sub>, and is unlikely to be Si limited (Caubet, Cornu, Saby, & Meunier, 2020). Two seeds per plant were sown in each pot and the largest plant was kept after germination. Pots of the N<sup>+ </sup>treatment received N four times during the experiment, for a total input of 0.94 g N pot<sup>-1</sup>, whereas pots of the N<sup>-</sup> treatment did not receive any N fertilisation. Plants were not protected from the rain, and were watered with amounts to avoid water excess or deficit. <em>Plant height, biomass and leaf [Si] measurements</em> Vegetative plant height, plant aboveground biomass and leaf [Si] were quantified at the beginning of the flowering stage. Vegetative height was measured as the distance between the soil surface and the tallest leaf without stretching the plant leaf. The leaf [Si] was quantified with an X-ray fluorescence spectrometer (Reidinger, Ramsey, & Hartley, 2012). Briefly, three most recent ligulate adult leaves were sampled on each individual, dried at 60°C for 72 h, and ball-milled (Retsch MM400 Mixer mill, Haan, Germany) for 3 min at a frequency of 20 Hz. Ground samples were pressed at 10 tons into pellets using a manual hydraulic press (Specac, Orpington, UK). Si analyses were performed using a Nitron XL3t900 GOLDD XRF analyser (Thermo Scientific, Winchester, UK). Silicon-spiked synthetic cellulose was used for calibration, and analyses were performed under helium atmosphere to avoid signal loss by air absorption (Reidinger et al., 2012). A reading was taken of each side of the pellet, approximately one hour apart, to account for <em>u</em>-drift in the instrument (Johnson, 2014). The concentration of Si in these three most recent ligulate adult leaves (in % of Si by dry weight) was considered to capture the intraspecific variation in leaf [Si] among the genotypes, the response to N fertilisation and plant-plant interactions, and potential relations between leaf [Si] and competition outcomes. Finally, all plant materials were harvested, dried at 60°C for 72 h, and weighed to obtain aboveground biomass. <em>Statistical analyses</em> <strong><em>Variation in leaf [Si] among genotypes, and response to N fertilisation </em></strong> Variation in leaf [Si] among the 19 wheat genotypes and their plasticity to N fertilisation were assessed only for the <em>single</em> plants to discriminate it from the neighbour effect. For both N treatments, differences in leaf Si across the 19 genotypes were tested by a one-way analysis of variance (ANOVA). To quantify the plasticity of leaf [Si] in response to N fertilisation among the 19 genotypes, we calculated log response-ratios (hereafter logRR) as the logarithm of ratios between individual trait values and corresponding genotype-mean values in N<sup>-</sup>, as follows: Differences in logRR among genotypes were tested by ANOVA, and genotype-mean logRR significantly different from zero were assessed with Student’s t-tests. A logRR below zero means that the treatment significantly decreased the trait values, while the opposite is true for logRR above zero. <strong><em>Plasticity to plant-plant interactions </em></strong> We first tested differences in leaf [Si] among the treatments <em>single</em>, <em>intra-</em> and <em>intergenotypic cultures</em> by ANOVA followed by <em>post hoc</em> tests using the ‘multcomp’ package (Hothorn, Bretz, & Westfall, 2008) for both N treatments. To quantify the plasticity of leaf [Si] to plant-plant interactions, we calculated logRR as the logarithm of ratios between individual trait values and corresponding genotype-mean values in <em>single</em>, independently for both N treatments. <em>Intra-</em> and <em>intergenotypic culture</em> treatments were considered either separately or pooled together as a global factor ‘plant-plant interactions’ to contrast single versus two-plants cultures in the analyses. Spearman rank correlation coefficients were calculated to test whether the ranking in genotype-mean logRR were conserved between both N treatments and between <em>intra-</em> and <em>intergenotypic cultures</em>. For the <em>intergenotypic culture</em> treatment, we further tested if neighbour identity influenced leaf [Si] by ANOVA, and for both N treatments. <strong><em>Relationships between leaf [Si], plant height and biomass </em></strong> We first tested differences in plant aboveground biomass and plant height across the different treatments (N and growth modality) by ANOVA, followed by <em>post hoc</em> tests. Relationships between aboveground biomass/plant height (dependent variables) and leaf [Si] (independent variable) were then tested through mixed-effect models with genotype identity as a random factor, using the package ‘nlme’ (Pinheiro, Bates, DebRoy, Sarkar, & Team, 2022). Models involving only the <em>single</em> individuals included both N treatments to test if a N-induced decrease in biomass affects leaf [Si], while models considering only plants with a neighbour were run separately for each N treatment. To test whether high-Si genotypes lost more or gain more biomass as a response to plant-plant interactions, we tested the significance of relationships between the logRR of plant biomass in response to plant-plant interactions and genotype-mean leaf [Si] in <em>single</em> by regression analyses, and for both N treatments. For each model, residuals were inspected visually to check assumptions. Appropriate variance structures were specified in a second model if required (Zuur, Ieno, Walker, Saveliev, & Smith, 2009). All analyses were conducted in the R environment (R Core Team, 2021).