Unbiased Estimation of Norway Spruce (Picea Abies L. Karst.) Chloroplast Structure: Heterogeneity Within Needle Mesophyll Under Different Irradiance and [Co2]

Abstract

The main objective of this study was to find out whether the selected chloroplast characteristics measured in the mesophyll layer nearest to the needle surface (i.e., the first mesophyll layer) could be representative for the whole needle cross section. Two chloroplast sampling approaches were applied on Norway spruce needles during the investigation of the effects of different levels of air CO2 concentration and irradiance: (i) sampling only from the first mesophyll layer, and (ii) systematic uniform random (SUR) sampling. The selected characteristics were: (i) chloroplast area, (ii) starch grain area, and (iii) starch areal density on median chloroplast cross sections, and (iv) chloroplast number per unit of needle volume. It was shown that the first mesophyll layer was not representative for estimating all evaluated characteristics except the chloroplast area. Sampling only there caused obtaining slightly biased results, while SUR sampling gave unbiased estimations at the cost of longer measuring time. The major effect of studied factors was in starch areal density and starch grain area, which were larger in sun needles in elevated CO2 concentration in comparison with sun needles in ambient CO2 concentration. In conclusion, it was demonstrated that the first layer of mesophyll is not always representative for the needle cross section. If technically feasible, SUR is recommended for analysis of chloroplast ultrastructure. The simplified sampling design can be applied, e.g., for comparisons of many different treatments. However, it should be combined with other approaches to characterize the chloroplast function and the results carefully considered and interpreted.

Modifications in Ultrastructural Characteristics and Redox Status of Plants under Environmental Stress: A Review

“…This approach enables a more accurate interpretation of results obtained from biochemical analysis, which sheds more light on plants' adaptive capacity. These helpful quantitative estimates of subcellular compartments by means of TEM have been utilized in numerous studies [160,[188][189][190][191][192][193][194] to achieve this goal. Increased size of chloroplasts, mitochondria, and plastoglobules [191] Increased number of chloroplasts and plastoglobules per 100 µm 2 visible field Decreased number of mitochondria per 100 µm 2 visible field High temperature (40 No significant changes in the cell area occupied by chloroplasts and in the size and length-to-width ratio of chloroplasts in the drought-resistant line [193] Significant reduction in the length-to-width ratios of chloroplasts and the cell area occupied by chloroplasts in drought-sensitive lines Needle-mesophyll cells Air CO 2…”

“…Increased number of chloroplasts per mesophyll volume (sampled systematically, uniformly, and randomly from the whole needle cross-section area) [194] Increased starch areal density and starch grain area in chloroplasts (sampled from both the whole needle cross-section area and from the first layer of mesophyll) Irradiance Increased starch areal density and starch grain area in chloroplasts (sampled from both the whole needle cross-section area and from the first layer of mesophyll)…”

A novel workflow for unbiased quantification of autophagosomes in 3D inArabidopsis thalianaroots

“…Stereological methods are – unfortunately – used much less frequently in plant science than in animal or human anatomy (for a review summarizing the application of stereology in plants, see [21]), although they were reported to outperform object counting in 2D sections [30]. Optical disector combined with the Cavalieri principle has recently been applied to determine the number of chloroplasts per volume of spruce needle or in single mesophyll cells [30,43], i. e. to count individual particles within a tissue which is very similar to the quantification of autophagosomes. That is why we consider stereological quantification a perfectly suitable tool for the assessment of the number of autophagosomes.…”