Computational protocol: Increased Needle Nitrogen Contents Did Not Improve Shoot Photosynthetic Performance of Mature Nitrogen-Poor Scots Pine Trees

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[…] The needles from the capacity and continuous measurements were harvested in August and November of 2013, respectively. The projected needle areas were determined using a flatbed scanner (Epson 1600+ equipped for dual scanning) and WinSEEDLE Pro 5.1a (Regent Instruments Inc., Quebec City, Canada) analysis software. The needle segments (20 mm) used in capacity measurements were cut along the edges of the tape holding them in place, and the needles used in the continuous measurements were detached from the shoot axis prior to scanning. The needle dry mass was determined after drying to constant weight at 70°C and an elemental analyzer (EA 1108, Fison Instruments, Rodano, Italy) was used to determine the mass-based needle N contents (Nm). Needle total Kjeldahl P contents were determined using Seal AutoAnalyzer HR 3 (Seal Analytical, Nordstedt, Germany; method G-189-97). The foliar nutrient contents of P. sylvestris exhibit some seasonality (Tamm, ; Näsholm and Ericsson, ). Therefore, Nm and the mass-based needle P contents (Pm) of the continuously measured needles, sampled later in the year, were expected to be greater than those of the needles used in the capacity measurements, although the two sets of needles likely had similar foliar nutrient contents in August. Furthermore, P. sylvestris needles tend to rotate around their axis and, thus, using whole needles for determining the leaf mass per projected needle area (LMA) results in greater estimates of LMA, Na, and Pa than analyses using shorter needle segments. This effect was quantified in a separate analysis that showed that LMA, Na, and Pa estimated for such segments were 12.5 ± 2.6% (n = 6) lower than for whole needles (Table ).In order to assess the within-canopy and age-related variation in arginine N, and its fraction of total needle N, an additional needle sampling on the studied trees was carried out in October 2014. Needles were collected from positions adjacent to those of the shoots used for capacity measurements the year before and stored at −20°C prior to analysis of their amino acid contents by reverse-phase liquid chromatography using a Waters Ultra High Performance (UPLC) system with a Waters Tunable UV detector (Waters, Corporation, Milford, MA, U.S.A.) following the procedure described in Inselsbacher et al. (). Needle N contents excluding arginine N were estimated assuming that the fractions of arginine N of total needle N at the studied canopy levels were equal in 2013 and 2014.The light climate of each shoot used in the photosynthetic capacity measurements is expressed as diffuse non-interceptance (openness) at the shoot location measured using LAI-2200 Plant Canopy Analyzer (Li-Cor Biosciences, Lincoln, Nebraska). The LAI-2200 was run in two sensor mode with one of the sensors placed above the canopy. The vertical variation in openness is shown in Supplementary material (Figure ). [...] Because the overall experimental design consisted of non-replicated plots centered on eddy-covariance flux measurement towers and due to the need of scaffolding towers for crown access, the measured trees were not randomly selected. While this raises concerns regarding pseudo-replication (Hurlbert, ) in response to potential between plots differences prior to the initial fertilizer application, the aim of the current experiment was to understand the photosynthetic constraints of P. sylvestris trees with strongly differing foliar N contents growing in the field under otherwise comparable environmental conditions rather than to study treatment effects on the stand-scale per se.All statistical analyses were carried out in IBM SPSS Statistics 20 (IBM Corporation, Armonk, NY, U.S.A.). A three-way repeated measures ANOVA (RMA) with needle age and canopy position as within-subjects factors and fertilizer treatment (plot) as a between-subjects factor was utilized to detect significant differences (P < 0.05) in needle photosynthetic, structural and chemical properties. Shapiro-Wilk's and Mauchly's tests were performed to test the data for normality and sphericity, respectively. When the sphericity assumption was met the factor effects were evaluated based on the multivariate tests (Wilks' lambda), and if this assumption was violated the degrees of freedom were corrected using Greenhouse-Geisser estimates of sphericity. In cases where the needle properties of 2-year-old needles were measured only in the upper canopy (see Tables , ), this age class was excluded from the analyses by three-way RMA. Separate tests with two-way RMA, with fertilizer treatment and age as factors, were used to evaluate the effects of needle age, including all three age classes, on the measured needle properties in the upper canopy. In cases where significant interactions between two factors were detected by RMA, tests of simple main effects were carried out to assess the responses to the individual factors. Bonferroni corrections were applied to post-hoc analyses of the effects of canopy position and needle age when appropriate.The photosynthetic capacity responses to needle nutrient contents were evaluated by regression analyses based on: (i)Na, (ii)Pa, (iii)Na and Pa, (iv) area-based needle N content excluding arginine N (Nax), and (v)Nax and Pa. When both N and P were included (i.e., cases iii and v), the co-dependencies of N and P were determined by multiple linear regression analysis that also included the N*P interaction term. Because fertilizer application was expected to affect the relationships between N, P and photosynthetic capacity parameters, the multiple regression analyses were carried out separately for each plot. Regression analyses were also used to investigate the between plots differences in the long-term shoot-scale photosynthetic performance responses to environmental variables and needle properties. The differences between the regression slopes and intercepts were evaluated using ANCOVA. If the relationship between the dependent and independent variables was non-linear data were log-log transformed prior to the ANCOVA analysis. […]

Pipeline specifications

Software tools WinSEEDLE, SPSS
Applications Miscellaneous, Macroscope & basic digital camera imaging
Diseases Deficiency Diseases, Phosphorus Metabolism Disorders
Chemicals Carbon, Nitrogen