Apple rootstocks affect functional leaf traits with consequential effects on carbon isotope composition and vegetative vigour.

Composite trees combine optimal traits from both the rootstock and the scion. Dwarfing rootstocks are commonly used to reduce shoot vigour and improve fruit quality and productivity. Although growth habits of different rootstocks have been clearly described, the underlying physiological traits affecting scion vigour are not well understood. Plant water status and stem water potential are strongly influenced by water supply and demand through the soil-plant-atmosphere continuum. In the scion, stomata regulate water loss and are essential to prevent hydraulic failure. Stomatal conductance influences leaf carbon isotope composition. Combined, the effects of reduced stomatal conductance and, consequently, carbon fixation may affect tree growth. These differences could also correspond to differences in scion vigour controlled by rootstock genotype. Here, vegetative growth, gas exchange, stem water potential and leaf δ13C were compared to determine how rootstocks affect scion water relations and whether these differences correspond to shoot vigour. There was a range in vigour among rootstocks by almost 2-fold. Net leaf carbon assimilation rates were lower in rootstocks with lower vigour. Rootstock vigour was closely associated with leaf gas exchange and stem water potential in the scion and was reflected in leaf δ13C signatures. Dwarfing was strongly affected by changes to plant water status induced by rootstock genotype and these changes are distinguishable when measuring leaf and stem δ13C composition. These observations indicate that scion water relations and leaf carbon isotope discrimination were affected by rootstock genotype. These results have implications for better understanding dwarfing mechanisms in apple rootstocks and the relationship with water-use traits.

Plant Uptake of Lactate-Bound Metals: A Sustainable Alternative to Metal Chlorides.

Global agricultural intensification has prompted investigations into biostimulants to enhance plant nutrition and soil ecosystem processes. Metal lactates are an understudied class of organic micronutrient supplement that provide both a labile carbon source and mineral nutrition for plant and microbial growth. To gain a fundamental understanding of plant responses to metal lactates, we employed a series of sterile culture-vessel experiments to compare the uptake and toxicity of five metals (Zn, Mn, Cu, Ni, and Co) supplied in lactate and chloride salt form. Additionally, primary root growth in plate-grown Arabidopsis thaliana seedlings was used to determine optimal concentrations of each metal lactate. Our results suggest that uptake and utilization of metals in wheat (Triticum aestivum L.) when supplied in lactate form is comparable to that of metal chlorides. Metal lactates also have promotional growth effects on A. thaliana seedlings with optimal concentrations identified for Zn (0.5-1.0 µM), Mn (0.5-1.0 µM), Cu (0.5 µM), Ni (1.0 µM), and Co (0.5 µM) lactate. These findings present foundational evidence to support the use of metal lactates as potential crop biostimulants due to their ability to both supply nutrients and stimulate plant growth.

Genotypic variation in nitrogen isotope discrimination in Populus balsamifera L. clones grown with either nitrate or ammonium.

Intraspecific variability in nitrogen use has not been comprehensively assessed in a natural poplar species. Here, a nitrogen isotope mass balance approach was used to assess variability in nitrogen uptake, assimilation and allocation traits in 25 genotypes from five climatically dispersed provenances of Populus balsamifera L. grown hydroponically with either nitrate or ammonium. Balsam poplar was able to grow well with either ammonium or nitrate as the sole nitrogen source. Variation within provenances exceeded significant provenance level variation. Interestingly, genotypes with rapid growth on nitrate achieved similar growth with ammonium. In most cases, the root:shoot ratio was greater in plants grown with ammonium. However, there were genotypes where root:shoot ratio was lower for some genotypes grown with ammonium compared to nitrate. Tissue nitrogen concentration was greater in the leaves and stems but not the roots for plants grown with ammonium compared to nitrate. There was extensive genotypic variation in organ-level nitrogen isotope composition. Root nitrogen isotope discrimination was greater under nitrate than ammonium, but leaf nitrogen isotope discrimination was not significantly different between plants on different sources. This can indicate variation in partitioning of nitrogen assimilation, efflux/influx (E/I) and root or leaf assimilation rates. The proportion of nitrogen assimilated in roots was lower under nitrate than ammonium. E/I was lower for nitrate than ammonium. With the exception of E/I, genotype-level variations in nitrogen-use traits for nitrate were correlated with the same traits when grown with ammonium. Using the nitrogen isotope mass balance model, a high degree of genotypic variation in nitrogen use traits was identified at both the provenance and, more extensively, the genotypic level.

Non-destructive Measurement of Calcium and Potassium in Apple and Pear Using Handheld X-ray Fluorescence.

Calcium and potassium are essential for cell signaling, ion homeostasis and cell wall strength in plants. Unlike nutrients such as nitrogen and potassium, calcium is immobile in plants. Localized calcium deficiencies result in agricultural losses; particularly for fleshy horticultural crops in which elemental imbalances in fruit contribute to the development of physiological disorders such as bitter pit in apple and cork spot in pear. Currently, elemental analysis of plant tissue is destructive, time consuming and costly. This is a limitation for nutrition studies related to calcium in plants. Handheld portable x-ray fluorescence (XRF) can be used to non-destructively measure elemental concentrations. The main objective was to test if handheld XRF can be used for semi-quantitative calcium and potassium analysis of in-tact apple and pear. Semi-quantitative measurements for individual fruit were compared to results obtained from traditional lab analysis. Here, we observed significant correlations between handheld XRF measurements of calcium and potassium and concentrations determined using MP-AES lab analysis. Pearson correlation coefficients ranged from 0.73 and 0.97. Furthermore, measuring apple and pear using handheld XRF identified spatial variability in calcium and potassium concentrations on the surface of individual fruit. This variability may contribute to the development of localized nutritional imbalances. This highlights the importance of understanding spatial and temporal variability in elemental concentrations in plant tissue. Handheld XRF is a relatively high-throughput approach for measuring calcium and potassium in plant tissue. It can be used in conjunction with traditional lab analysis to better understand spatial and temporal patterns in calcium and potassium uptake and distribution within an organ, plant or across the landscape.

Variation in fluxes estimated from nitrogen isotope discrimination corresponds with independent measures of nitrogen flux in Populus balsamifera L.

Acquisition of mineral nitrogen by roots from the surrounding environment is often not completely efficient, in which a variable amount of leakage (efflux) relative to gross uptake (influx) occurs. The efflux/influx ratio (E/I) is, therefore, inversely related to the efficiency of nutrient uptake at the root level. Time-integrated estimates of E/I and other nitrogen-use traits may be obtainable from variation in stable isotope ratios or through compartmental analysis of tracer efflux (CATE) using radioactive or stable isotopes. To compare these two methods, Populus balsamifera L. genotypes were selected, a priori, for high or low nitrogen isotope discrimination. Vegetative cuttings were grown hydroponically, and E/I was calculated using an isotope mass balance model (IMB) and compared to E/I calculated using (15) N CATE. Both methods indicated that plants grown with ammonium had greater E/I than nitrate-grown plants. Genotypes with high or low E/I using CATE also had similarly high or low estimates of E/I using IMB, respectively. Genotype-specific means were linearly correlated (r = 0.77; P = 0.0065). Discrepancies in E/I between methods may reflect uncertainties in discrimination factors for the assimilatory enzymes, or temporal differences in uptake patterns. By utilizing genotypes with known variation in nitrogen isotope discrimination, a relationship between nitrogen isotope discrimination and bidirectional nitrogen fluxes at the root level was observed.

Nitrogen isotope discrimination as an integrated measure of nitrogen fluxes, assimilation and allocation in plants.

Fractionation of nitrogen isotopes between a plant and its environment occurs during uptake and assimilation of inorganic nitrogen. Fractionation can also occur between roots and the shoot. Under controlled nitrogen conditions, whole-plant and organ-level nitrogen isotope discrimination (Δ(15) N) is suggested to primarily be a function of three factors: nitrogen efflux back to the substrate relative to gross influx at the root (efflux/influx), the proportion of net influx assimilated in the roots and the export of remaining inorganic nitrogen for assimilation in the leaves. Here, an isotope discrimination model combining measurements of δ(15) N and nitrogen content is proposed to explain whole-plant and organ-level variation in δ(15) N under steady-state conditions and prior to any significant retranslocation. We show evidence that nitrogen isotope discrimination varies in accordance with changes to nitrogen supply or demand. Increased whole-plant discrimination (greater Δ(15) N or more negative δ(15) N relative to the source nitrogen δ(15) N) indicates increased turnover of the cytosolic inorganic nitrogen pool and a greater efflux/influx ratio. A greater difference between shoot and root δ(15) N indicates a greater proportion of inorganic nitrogen being assimilated in the leaves. In addition to calculations of integrated nitrogen-use traits, knowledge of biomass partitioning and nitrogen concentrations in different plant organs provides a spatially and temporally integrated, whole-plant phenotyping approach for measuring nitrogen-use in plants. This approach can be used to complement instantaneous cell- and tissue-specific measures of nitrogen use currently used in nitrogen uptake and assimilation studies.

Quantifying remobilization of pre-existing nitrogen from cuttings to new growth of woody plants using 15N at natural abundance.

BACKGROUND: For measurements of nitrogen isotope composition at natural abundance, carry-over of pre-existing nitrogen remobilized to new plant growth can cause deviation of measured isotope composition (δ15N) from the δ15Nof newly acquired nitrogen. To account for this problem, a two-step approach was proposed to quantify and correct for remobilized nitrogen from vegetative cuttings of Populus balsamifera L. grown with either nitrate (δ15N = 58.5‰) or ammonium (δ15N = -0.96‰). First, the fraction of carry-over nitrogen remaining in the cutting was estimated by isotope mass balance. Then measured δ15N values were adjusted for the fraction of pre-existing nitrogen remobilized to the plant. RESULTS: Mean plant δ15N prior to correction was 49‰ and -5.8‰ under nitrate and ammonium, respectively. Plant δ15N was non-linearly correlated to biomass (r2 = 0.331 and 0.249 for nitrate and ammonium, respectively; P < 0.05) where the δ15N of plants with low biomass approached the δ15N of the pre-existing nitrogen. Approximately 50% of cutting nitrogen was not remobilized, irrespective of size. The proportion of carry-over nitrogen in new growth was not different between sources but ranged from less than 1% to 21% and was dependent on plant biomass and, to a lesser degree, the size of the cutting. The δ15N of newly acquired nitrogen averaged 52.7‰ and -6.4‰ for nitrate and ammonium-grown plants, respectively; both lower than their source values, as expected. Since there was a greater difference in δ15N between the carried-over pre-existing and newly assimilated nitrogen where nitrate was the source, the difference between measured δ15N and adjusted δ15N was also greater. There was no significant relationship between biomass and plant δ15N with either ammonium or nitrate after adjusting for carry-over nitrogen. CONCLUSION: Here, we provide evidence of remobilized pre-existing nitrogen influencing δ15N of new growth of P. balsamifera L. A simple, though approximate, correction is proposed that can account for the remobilized fraction in the plant. With careful sampling to quantify pre-existing nitrogen, this method can more accurately determine changes in nitrogen isotope discrimination in plants.

Whole-plant and organ-level nitrogen isotope discrimination indicates modification of partitioning of assimilation, fluxes and allocation of nitrogen in knockout lines of Arabidopsis thaliana.

The nitrogen isotope composition (δ¹5N) of plants has potential to provide time-integrated information on nitrogen uptake, assimilation and allocation. Here, we take advantage of existing T-DNA and γ-ray mutant lines of Arabidopsis thaliana to modify whole-plant and organ-level nitrogen isotope composition. Nitrate reductase 2 (nia2), nitrate reductase 1 (nia1) and nitrate transporter (nrt2) mutant lines and the Col-0 wild type were grown hydroponically under steady-state NO3⁻ conditions at either 100 or 1000 µM NO3⁻ for 35 days. There were no significant effects on whole-plant discrimination and growth in the assimilatory mutants (nia2 and nia1). Pronounced root vs leaf differences in δ¹5N, however, indicated that nia2 had an increased proportion of nitrogen assimilation of NO3⁻ in leaves while nia1 had an increased proportion of assimilation in roots. These observations are consistent with reported ratios of nia1 and nia2 gene expression levels in leaves and roots. Greater whole-plant discrimination in nrt2 indicated an increase in efflux of unassimilated NO3⁻ back to the rooting medium. This phenotype was associated with an overall reduction in NO3⁻ uptake, assimilation and decreased partitioning of NO3⁻ assimilation to the leaves, presumably because of decreased symplastic intercellular movement of NO3⁻ in the root. Although the results were more varied than expected, they are interpretable within the context of expected mechanisms of whole-plant and organ-level nitrogen isotope discrimination that indicate variation in nitrogen fluxes, assimilation and allocation between lines.