Monitoring of Daily Training Load and Training Load Responses in Endurance Sports: What do Coaches Want?

Accurate assessment of training load (TL) and training load responses (TLR) might be useful for an optimized training regulation and prevention of overtraining. No consensus on a gold-standard for measuring TL or intensity in endurance sports has been reported in the available literature so far. The aim of the present article is i) to identify feasible parameters to measure TL and TLR in daily training and ii) to compare these scientific approaches with the needs of elite endurance coaches. Therefore, the first part provides a systematic review of the current literature and the second part concentrates on the results of a questionnaire that assessed the coaches’ requirements for monitoring daily endurance training. The systematic review revealed that the combination of both quantitative and qualitative data seems most promising to evaluate TL and TLR. Thus, validated questionnaires or rating of perceived exertion (RPE), combined with physiological parameters, such as heart rate, are often used and seem to provide the most reliable results. From the coaches’ perspective, duration and kind of training, RPE, as well as personal remarks in the athletes’ training diaries are considered to be essential information. Further, the coaches favor a feasible system that collects large amounts of directly measurable and perceived data and that is able to learn from previous events in order to present the most important information in a short individual overview. When comparing both parts of the present study, it becomes clear that the scientific research cannot yet fully respond to the coaches’ requests, however, based on the coaches’ propositions, scientific research might be stimulated to tackle this challenge in the near future

A Holistic Approach to Analyze Systemic Jasmonate Accumulation in Individual Leaves of Arabidopsis Rosettes Upon Wounding

Phytohormones, especially jasmonates, are known to be mediators of the plant responses to wounding and herbivore feeding. Their role in such stress responses has been largely studied locally in treated leaves. However, less is known about the induced systemic distribution of phytohormone signals upon these kinds of stresses. Here, a holistic approach was performed in order to investigate the systemic phytohormone pattern in the rosette of Arabidopsisthaliana after herbivore-related wounding. Levels of different stress-related phytohormones such as jasmonates, abscisic acid, and salicylic acid were analyzed in individual leaves. We demonstrate that the typically used sampling method, where leaves are first cut and immediately frozen, causes false-positive results since cutting already induces systemic jasmonate elevations within less than 1.6 min. Therefore, this approach is not suitable to study systemic phytohormone changes in the whole plant. By developing a new method where leaves are frozen first and subsequently cut, sampling-induced phytohormone elevations could be reduced. Using this new method, we show that jasmonic acid and its active isoleucine conjugate (jasmonoyl-isoleucine) are involved in the fast systemic wound response of Arabidopsis. A systemic induction of the jasmonates’ precursor, 12-oxo-phytodienoic acid, was not observed throughout our treatments. The systemic phytohormone distribution pattern is strongly linked to the vascular connections between the leaves, providing further evidence that the vascular system is used for long distance-signaling in Arabidopsis. Besides already known vascular connections, we also demonstrate that the systemic distribution of jasmonate signals can be extended to distant leaves, which are systemically but indirectly connected via another vascularly connected leaf. This holistic approach covering almost the whole Arabidopsis rosette introduces a method to overcome false-positive results in systemic phytohormone determinations and demonstrates that wounding-induced long-distance signaling includes fast changes in jasmonate levels in systemic, non-treated leaves

Herbivory-responsive calmodulin-like protein CML9 does not guide jasmonate-mediated defenses in <i>Arabidopsis thaliana</i>

<div><p>Calcium is an important second messenger in plants that is released into the cytosol early after recognition of various environmental stimuli. Decoding of such calcium signals by calcium sensors is the key for the plant to react appropriately to each stimulus. Several members of Calmodulin-like proteins (CMLs) act as calcium sensors and some are known to mediate both abiotic and biotic stress responses. Here, we study the role of the <i>Arabidopsis thaliana</i> CML9 in different stress responses. CML9 was reported earlier as defense regulator against <i>Pseudomonas syringae</i>. In contrast to salicylic acid-mediated defense against biotrophic pathogens such as <i>P</i>. <i>syringae</i>, defenses against herbivores and necrotrophic fungi are mediated by jasmonates. We demonstrate that <i>CML9</i> is induced upon wounding and feeding of the insect herbivore <i>Spodoptera littoralis</i>. However, neither different <i>CML9</i> loss-of-function mutant lines nor overexpression lines were impaired upon insect feeding. No difference in herbivore-induced phytohormone elevation was detected in <i>cml9</i> lines. The defense against the spider mite <i>Tetranychus urticae</i> was also unaffected. In addition, <i>cml9</i> mutant lines showed a wild type-like reaction to the necrotrophic fungus <i>Alternaria brassicicola</i>. Thus, our data suggest that CML9 might be a regulator involved only in the defense against biotrophic pathogens, independent of jasmonates. In addition, our data challenge the involvement of CML9 in plant drought stress response. Taken together, we suggest that CML9 is a specialized rather than a general regulator of stress responses in <i>Arabidopsis</i>.</p></div

Genetic differences between <i>cml9-a</i> and <i>cml9-b</i>.

<p>(<i>a</i>) Schematic overview of <i>CML9</i> with T-DNA insertions and the used primers for RT- and qRT-PCR. Exons are indicated with E, introns with I. Light gray triangles indicate T-DNA insertions. RT primers used are indicated by black arrows, qRT primers by grey arrows (qRT FP1 and qRT RP1 are published as CML9 primers in Vadassery, Scholz [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197633#pone.0197633.ref016" target="_blank">16</a>]). Total length of <i>CML9</i> gDNA without insertions is 1137 bp. (<i>b</i>) Verification of T-DNA insertions in <i>CML9</i> by genotyping. The expected product length is indicated on the right sites of the respective pictures. (<i>c</i>) Semi quantitative RT-PCR analysis of <i>CML9</i> expression in wild type and knock-out mutants. Plants were treated with a pattern wheel and either water (WW) or <i>S</i>. <i>littoralis</i> oral secretion (WOS) was applied for 30 min. Untreated plants were used as controls (CON). Besides full length expression, expression of the E1-E3 fragment, upstream of the intronic T-DNA insertions, is shown. Expression of <i>ACTIN</i> was used as quantitative control. The expected product length is written on the right sites of the respective pictures. Asterisks indicate unspecific bands in the <i>cml9-a</i> mutant. (<i>d</i>) Normalized fold expression of <i>CML9</i> E1-E3 fragment in wild type and <i>cml9-a</i> and <i>cml9-b</i> lines. Plants were treated as described in (<i>c</i>). Expression level was normalized with respect to the <i>RPS18B</i> transcript level. Bars represent the means ± SE (n ≥ 5). Experiments were repeated two times independently. Statistically significant changes were determined by two-way ANOVA. Results of statistical analysis are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197633#pone.0197633.t001" target="_blank">Table 1</a>.</p

Response of <i>A</i>. <i>thaliana</i> wild type and <i>cml9</i> lines to <i>A</i>. <i>brassicicola</i> infection.

<p>Macroscopic observation of lesion formation (<i>a</i>) and measurement of chlorophyll fluorescence (<i>b</i>) of wild type and mutant leaves 3 and 4 day post-inoculation (dpi) with <i>A</i>. <i>brassicicola</i> spore suspension (Ab) or 0.01% Tween-20 solution as mock (M). Experiment was repeated three times independently. Plants shown are representative. Statistically significant differences in chlorophyll fluorescence of different genotypes among one treatment were determined by one-way ANOVA. No significant differences were measured, as indicated by the letters.</p

Phytohormone contents of <i>A</i>. <i>thaliana</i> wild type and <i>cml9</i> mutant plants after <i>S</i>. <i>littoralis</i> feeding.

<p>Levels of <i>cis</i>-OPDA (<i>a</i>), JA (<i>b</i>), JA-Ile (<i>c</i>), SA (<i>d</i>), ABA (<i>e</i>) after larval feeding for one week in ng g^-1 fresh weight (FW). Phytohormones were extracted only from local fed leaves. Untreated plants were used as controls. Bars represent means ± SE. Experiment was repeated three times independently (n ≥ 13). Statistically significant differences between phytohormone content of different genotypes among one treatment were determined by Kruskal-Wallis one-way ANOVA on ranks, using Dunn’s method as post-hoc test. No significant differences were measured, as indicated by the letters. Legend for color code see (<i>a</i>).</p

Susceptibility of different <i>Arabidopsis</i> mutant lines of <i>CML9</i> on <i>S</i>. <i>littoralis</i> feeding.

<p>Gain of larval weight was determined after feeding on Col-0 wild type plants and <i>cml9-a</i> and <i>cml9-b</i> knock-out lines (<i>a</i>), Col-8 and Ws-4 wild type plants and <i>cml9-1</i> and <i>cml9-2</i> knock-out lines (<i>b</i>) and Col-8 wild type plants and OE-CC-2 and OE-CC-5 overexpression lines (<i>c</i>) for one week. First instar larvae of <i>S</i>. <i>littoralis</i> were pre-weighed to reduce experimental variation. Three larvae were placed on each plant. After feeding period larval weight was determined. The boxplots show the distribution of the measured data. The box indicates the middle 50% of the data points. Black triangles represent outliers and the black squares the mean values. Whiskers are defined as 1.5 fold interquartile range (IQR). Experiments were repeated at least five times independently (n = 134 (Col-0), n = 133 (<i>cml9-a</i>), n = 139 (<i>cml9-b</i>), n = 98 (Col-8), n = 92 (<i>cml9-1</i>), n = 109 (Ws-4), n = 111 (<i>cml9-2</i>), n = 89 (OE-CC-2), n = 91 (OE-CC-5)). Statistically significant differences between larval weights of different genotypes were determined by unpaired two-sample Wilcoxon test. Asterisk indicates significance (* P < 0.05), n.s. means not significant.</p

Expression of <i>CML9</i> in response to different herbivore-associated stimuli.

<p>Changes in <i>CML9</i> transcript level in <i>A</i>. <i>thaliana</i> wild type leaves (Col-0) upon <i>S</i>. <i>littoralis</i> feeding (<i>a</i>), mechanical wounding with a pattern wheel and application of either water or <i>S</i>. <i>littoralis</i> OS (<i>b</i>), mechanical damage by MecWorm (<i>c</i>) or ABA spray combined with MecWorm treatment (<i>d</i>) are plotted. Expression level in (<i>a</i>), (<i>b</i>) and (<i>c</i>) was determined after 30, 60, 90, 120 and 180 min of treatment. Untreated plants were used as controls. Fold expression in (<i>d</i>) was measured after 30 min of MecWorm treatment with a 60 min pre-incubation with 100 μM ABA solution or a 0.02% ethanol solution, or just incubation with ABA. Control plants were treated with 0.02% ethanol. The <i>CML9</i> fold expression was normalized with respect to the <i>RPS18B</i> transcript level and calculated relative to respective controls. Bars represent the means ± standard error (SE) (n ≥ 6 (<i>a</i>), n ≥ 5 (<i>b</i>), n ≥ 11 (<i>c</i>), n ≥ 10 (<i>d</i>)). Experiments were repeated at least two times independently. Statistically significant changes in expression levels were determined by one-sample Wilcoxon test (<i>a</i>) or one-sample t-test (<i>c</i>). Statistically significant differences between the different treatments were determined by unpaired two-sample Wilcoxon test at each time point separately (<i>b</i>) or by one way ANOVA (<i>d</i>). Asterisks indicate significances (* P < 0.05, *** P < 0.001). P-values for (<i>a</i>), (<i>b</i>) and (<i>c</i>) are FDR corrected. Letters in (d) indicate no statistic differences.</p

Comparison of drought stress response of wild type and <i>cml9</i> mutants.

<p>Representative pictures (<i>a</i>) and mean ABA level ± SE (<i>b</i>) of wild type, <i>cml9-a</i> and <i>cml9-b</i> before (0 d) and after drought (11 d and 18 d). Experiment was started with 4-week-old plants (0 d). Plants exposed to drought for 18 d were watered once after 11 d. All plants shown in (<i>a</i>) are independent from each other. Treatment was repeated 4 times independently (n = 20). Statistically significant differences between ABA content of different genotypes among one treatment were determined by one-way ANOVA, using Student-Newman-Keuls (SNK) method as post-hoc test. Different letters indicate significant changes (P < 0.05).</p