# Unsupervised analysis of NIRS spectra to assess complex plant traits: leaf senescence as a use case

Héloïse Villesseche, Martin Ecarnot, Elsa Ballini, Ryad Bendoula, Nathalie

Gorretta, Pierre Roumet

► To cite this version:

Héloïse Villesseche, Martin Ecarnot, Elsa Ballini, Ryad Bendoula, Nathalie Gorretta, et al. Unsupervised analysis of NIRS spectra to assess complex plant traits: leaf senescence as a use case. Plant Methods, 2022, 18 (1), pp.100. 10.1186/s13007-022-00927-6. hal-03773243

HAL Id: hal-03773243

https://hal.inrae.fr/hal-03773243

Submitted on 9 Sep 2022

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH

Open Access

<figure><img src="image_2.png" /></figure>

Check for
updates

# Unsupervised analysis of NIRS spectra to assess complex plant traits: leaf senescence as a use case

Héloïse Villesseche<fnref n="1" />*, Martin Ecarnot<fnref n="1" />, Elsa Ballini², Ryad Bendoula³, Nathalie Gorretta<fnref n="1" /> and Pierre Roumet<fnref n="1" />

**Abstract**

**Background:** As a rapid and non-destructive method, Near Infrared Spectroscopy is classically proposed to assess plant traits in many scientific fields, to observe enlarged genotype panels and to document the temporal kinetic of some biological processes. Most often, supervised models are used. The signal is calibrated thanks to reference measurements, and dedicated models are generated to predict biological traits. An alternative unsupervised approach considers the whole spectra information in order to point out various matrix changes. Although more generic, and faster to implement, as it does not require a reference data set, this latter approach is rarely used to document biological processes, and does requires more information of the process.

**Methods:** In our work, an unsupervised model was used to document the flag leaf senescence of durum wheat (Triticum turgidum durum). Leaf spectra changes were observed using Moving Window Principal Component Analysis (MWPCA). The dates related to earlier and later spectra changes were compared to two key points on the senescence time course: senescence onset (T0) and the end of the leaf span (T1) derived from a supervised strategy.

**Results:** For almost all leaves and whatever the signal pre-treatments and window size considered, the MWPCA found significant spectral changes. The latter was highly correlated with T1 (0.59 ≤ r ≤ 0.86) whereas the correlations between the first significant spectrum changes and T0 were lower (0.09 ≤ r ≤ 0.56). These different relationships are discussed below since they define the potential as well as the limitations of MWPCA to model biological processes.

**Conclusion:** Overall, our study demonstrates that the information contained in the spectra can be used when applying an unsupervised method, here the MWPCA, to characterize a complex biological phenomenon such leaf senescence. It also means that using whole spectra may be relevant in agriculture and plant biology.

**Keywords:** NIR, Leaf senescence, Unsupervised model, Wheat, Temporal kinetic

**Background**

The plant phenotype, i.e., the set of observable characteristics of an individual, reflects both the gene expression (genotype) and the amount of available resources (mineral nutrients, water, temperature, light) as well their interaction. Phenotypic measurements are essential to

understand how organisms interact with their environments to identify, for example, the genetic determinism involved in local adaptation, or to achieve a more sustainable production in agronomy. Too often, measurements of most of these traits require destructive and time costly methods, limiting extended observations of numerous individuals and/or along temporal series. The development of methodologies to assess an enlarged list of these traits is of great interest for many fields of study: agronomy, ecology, genetics and phytopathology. For two decades, numerous applications based on non-invasive and

<footnote>*Correspondence: heloise.villesseche@inrae.fr</footnote>

<footnote n="1">¹ AGAP, CIRAD, INRAE, Institut Agro, Univ Montpellier, Montpellier, France
Full list of author information is available at the end of the article</footnote>
non-destructive methodologies, such as imaging analysis or near infrared spectroscopy, have been developed to infer trait values [1, 2]. As most of them are complex traits resulting from numerous and successive processes as well as their interactions, two main complementary strategies have been proposed to phenotype these traits.

The first one leads to the dissection of these complex traits into elementary traits to facilitate the identification of underlying physiological pathways or genetic determinism. This strategy is commonly used in scientific communities such as physiologists or geneticists for whom a better understanding of the building of complex traits is essential. As most often the elementary traits are not easily visually observable, extensive works related to the use of some non-destructive and non-invasive methods, largely based on the use of visible and near infrared (VIS-NIR) imaging and spectroscopy, have been undertaken [3]. Thanks to supervised models based on linear or nonlinear signal treatment methods, the calibrations provided relevant predictions with documented accuracy and robustness which can be used as proxies of the key traits targeted [4, 5]. The second strategy directly targets the complex trait to document some changes, or to detect some possible discrepancies along its time course; this global approach is frequently used in a wide range of disciplines such as transformation process monitoring, agronomy and phytopathology [6]. Both strategies are largely supported by the development of high throughput phenotyping platforms.

Overall, if rapid and non-destructive phenotyping methods such as VIS-NIR spectroscopy are highly desirable to document some changes in traits during their time course, both supervised and unsupervised methods—which do not require any signal calibration—can be used. Indeed, according to the hypothesis that a VIS-NIR spectra signal is related to the global status of the biological matrix at a given time, non-supervised models based on raw spectra information gathered along the process are pertinent when documenting some matrix changes during its time course, providing some alerts [7]. Different unsupervised methods have been proposed to document matrix changes but all follow the same principle: no calibration is made on the data set. The idea is to find common characteristics between spectra and to find hidden patterns indicating the presence or absence of change in the matrix. Most of the unsupervised strategies are based on the Principal Component Analysis (PCA). Among them, Moving Window Principal Components Analysis (MWPCA) has been developed to accurately pinpoint small changes in process monitoring [8].

Leaf senescence, which is the final stage of leaf development, is one example of these complex biological processes. Besides chlorophyll degradation which is well

known, numerous additional molecular and biochemical processes contribute to the senescence syndrome [9, 10]. When a leaf switches its developmental program from maturation to senescence, many leaf mobile nutrients involved in different pathways, such as carbon acquisition, are degraded and recycled for re-use in upper and still growing parts of the plant, e.g. younger leaves or in the forming reproductive organs. As the content of many biochemical components drop significantly during senescence [11], this process can be documented through many traits: leaf green area losses, leaf water content, decline of photosynthetic capacities [12] or a decrease of leaf biochemical content such as chlorophyll, nitrogen or water [13]. Whatever the trait considered, the senescence process follows a similar temporal pattern: after a lag phase during which the leaf maintains its physiological activity at a constant level, the content of biochemical components or enzymatic activity drops dramatically to reach a second plateau with minimal values. On cereals and particularly on wheat the timing of the onset and the end of senescence are considered as key points when comparing genotype senescence properties [14–16], as well as targeted physiological traits to predict grain yield since positive correlations have been reported between delayed senescence and grain yield both under limiting and non-limiting conditions [17, 18].

Based on spectra reflectance, numerous supervised models have been extensively used to generate some proxies of these traits to facilitate the collection of phenotype data sets. However these components have different time courses and they interact with the amount of resources available (nutrients and water in particular), demonstrating different senescence dynamics and an environmental dependent response [5]. These studies have shown that there is an intra specific variability in the onset and rate of senescence (for example see [16, 19, 20]). The onset of senescence is a complex trait that illustrates the transition of the leaf when grain N demand exceeds soil N uptake, generating translocation of N from stems and leaves and affecting carbon assimilation [21]. A delay in this onset or a lower rate of senescence will have a direct impact on wheat yield [5, 22–24].

The idea that the spectral signature could be used directly to characterize the presence and the state of vegetation was proposed following the development of the early sensors which had low spectral resolution [18, 25]. The first use of a spectral index, the normalized difference vegetation index (NDVI) was described by Rouse et al. [11] to document vegetation in the central region of the United States. Nevertheless, important limitations were reported for the use of NDVI to document the leaf senescence process. Only two wavelengths have been used to compute the index, NDVI is focused on
chlorophyll content, which turned out to be non-relevant to physiological changes occurring during senescence onset [26]. To avoid some confounding effects and to properly assess leaf senescence, the use of a higher number of bands made possible by a better spectral resolution device coupled with a supervised model was proposed [4]: visual senescence scoring was used as reference measurements and calibration of spectra data was performed to fit the senescence dynamics during the grain filling period. Here, the use of the supervised approach can be debated. Firstly, the reference analysis was based on visual information; as this measurement was neither objective nor precise, the generated background noise affected the accuracy of the model; secondly, the validity domain of this calibration is uncertain, thus limiting its use to new data sets. Thus, regarding the complexity of the senescence process and the limits of supervised models, an alternative approach based on an unsupervised model such as MWPCA where it is postulated that some changes occurring over time in a spectral data set are a signature of the senescence process seems promising.

In order to establish the limits of the unsupervised approach, we have compared the time course of flag wheat leaf senescence using both supervised and unsupervised models based on spectral reflectance. Large range of senescence time courses was generated thanks to the genotype sampling and mineral nutrition availability.

# Materials and methods

## Plant material

A total of 113 F<sub>8</sub> Recombinant Inbred Lines (RILs) from six crosses involving four durum wheat cultivars (Néo-dur, Primadur, Lloyd and Ixos) were used in the present study since they represented a high variability for senescence time course parameters.

## Experimental conditions

The trial has been conducted on durum wheat (*Triticum turgidum durum*) in greenhouse at the DiasCope experimental unit from Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAe, Mauguio, France, 43°37' N, 4° E) in 2011. Plants were sown on 5 Jan. 2011 in Jiffy peat pellets. After vernalization in a controlled chamber (4 °C for 5 weeks) they were transferred to a greenhouse (15 Feb. 2011) and grown in vermiculite in 1.2 L pots (one plant per pot). To maximize the variability range of flag leaf senescence, plants have been cultivated under two types of nitrogen fertilization conditions and observed daily from flowering to maturity. In the first nitrogen treatment, 104 plants (one plant/RILs), were irrigated once daily with 5 mM KNO3 until anthesis and with 1 mM KNO3 from anthesis to grain maturity; in the second one, 35 plants (representing

35 different RILs) were irrigated once daily with 1 mM KNO3 until anthesis and then with 5 mM KNO3 for 550 Growing Degree-Day (-GDD-) after heading. Potassium concentrations were adjusted for treatments with 1 mM KNO3 by adding 4 mM KCl to the nutrient solution. Irrigation was stopped when the grain reached physiological maturity. Greenhouse temperature was regulated by a cooling system and the opening and closing of greenhouse vents and was measured with independent sensors. The walls of the greenhouse were whitewashed to standardize the amount of light received by each plant. Further information about the settings of the experiment are described in Vilmus et al. [27].

## Spectra acquisitions

For each plant, all observations were made daily on the last leaf (flag leaf) from flowering to physiological maturity (stages Z65 and Z85 from Zadoks scale); they were numbered from one (for the first at flowering) to n (the last at physiological maturity). Observations were made in a non-destructive manner with a LabSpec 2500 portable Vis/Nir spectrometer, which has a spectral range between 350 and 2500 nm with a spectral resolution of 3 nm between 350 and 1000 nm to 10 nm between 1000 and 2500 nm (Analytical Spectral Devices, Inc., Boulder, CO, USA) using the LeafClip and a white background panel, allowing us to obtain transmittance spectra [28]. Each day, two measurements were performed by dividing the flag leaf into three equal parts along its length, with the first measurement made on the proximal third and the second on the distal third.

Based on these spectra data two different analysis were performed; firstly, a classical supervised strategy was used: calibrations were made to document the biochemical properties of the flag leaf during senescence process; secondly, whole spectra information (raw or pre-processed spectra data) were used in an unsupervised strategy to document the evolution of the leaf main features.

## Supervised strategy: NIR calibration development and temporal kinetic modeling

In a first step, quantitative calibrations from whole spectra were made to assess three biochemical leaf parameters involved in leaf senescence from data spectra, namely nitrogen, chlorophyll and water content. To carry out these calibrations, a small but representative group of samples were selected to build the models, and these calibrations were applied on all other measured spectra. A common protocol was applied for the three calibrations. First, spectra were taken, as described above, on leaves that were analyzed with a specific reference method for each parameter. Then calibrations were performed with Partial Least Square regression [29], using random
repeated k-fold cross-validation to optimize the number of latent variables of the model, and to compute model-fitting statistics (standard error of cross validation, coefficient of determination). For more details, please refer to the Additional file 1: Table S1.

In a second step, logistic regression was used to fit the temporal evolution of these components from flowering to the physiological maturity where time was expressed in cumulative GDD based on average daily temperature. For each leaf, the model is estimated as in Eq. (1):

$$f(x) = d + \frac{a}{1 + b * e^{c*x}} \qquad (1)$$

where a, b, c and d are parameters allowing the adjustment of the sigmoid curve for each individual and x being the date given in GDD. The results extracted from the model are the T0 and T1, corresponding respectively to the end of the first plateau and the end of the drop of the sigmoid curve. Here, these two key points have been considered as the onset and the end of the senescence. For each flag leaf, T0 and T1 have been computed for each of the three types of biochemical components according to the following formulae:

$$T_0 = d + \frac{a(1 - \gamma)}{1 + e^b} \quad \text{and} \quad T_1 = d + \frac{a\gamma}{1 + e^b} \qquad (2)$$

where a, b and d are three parameters of the sigmoid curve (Eq. 1) and γ =0.01.

Model evaluation was made using the efficiency calculated as in Eq. 3:

$$Efficiency = 1 - \frac{\sum_{1}^{i} (y || i - f(x_i))}{\sum_{1}^{i} (y || i - mean(y))} \qquad (3)$$

where i is varying from 1 to the number of dates taken in the time-series for each individual and y is the result of f(x). This model is described in detail in Vilmus et al. [27].

## Unsupervised strategy: Moving Window Principal Component Analysis (MWPCA)

A MWPCA [8] based approach was proposed here to detect changes that could lead the process to dysfunction, and then give an early alarm. Spectra collected at the successive dates from flowering to physiological maturity represented data variation. An abnormal deviation of spectra from those collected before could be considered as a major change in leaf features. To begin, Principal Component Analysis (PCA) was calibrated on training data representing the initial leaf state. From this model, the covariance and loading matrix P were computed. Secondly, new observations were projected onto the reference PCA model; a T² metric was used to

quantify spectra changes at t+1. The new observation xi is centered, using the parameters of the training data.

The initial PCA model was calibrated for each leaf, considering a spectra data matrix X (n × p) with n the number of observations and p the number of spectral variables (Eq. 4):

$$X_{(n \times p)} = T_{(n \times a)} \cdot P_{(a \times p)}^{T} + E_{(n \times p)} \qquad (4)$$

T is the score's matrix with a (a=1, 2 ...) the number of retained Principal Components (PCs), P^T is the trans-pose of loadings matrix which explains the relationship between spectral variables and PCs and E is the residual matrix. Before implementing PCA, variables are centered. PCA control charts are based on the Hotelling's T² statistics, being the squared Mahalanobis distance of the spectra x in the PCA model subspace [8] and calculated according to Eq. 5:

$$T^2 = x_i P \Lambda_a^{-1} P^T x_i^T \qquad (5)$$

where ∧ is the covariance matrix of the a scores column and xi is the i-th observation predicted through the PCA model. To define the change in the time-series, the hypothesis is made that the Hotelling's T² is following a normal law. Based on this hypothesis, a significant change is supposed to be a T² out of the normal distribution, modeled by the normal law. Significant changes will not be excluded from the next window, because this “new state” of the process will affect the next observations and could even result in a new normal steady state.

The adopted algorithm of the MWPCA is shown in detail in Fig. 1.

In MWPCA, the basic algorithm stipulates that a data window of fixed length be moved in real time to update the PCA model once a new sample is available. With each new observation, this window excludes the oldest observation and includes the newest one. One challenge in implementing MWPCA is to select the window length (also called window size). Due to the number of our dataset observations (~30 dates), we decided to test the influence of the window size on our dataset. During the analysis, windows sized between 3 and 15 were tested, equivalent to taking from 3 to 15measurements to build the core spectra to refer in the initial foliar stage. The number of components was also updated to correspond to the maximum number of components possible according to the window size. Knowing that two spectra of each plant were taken every day, the maximum number of components corresponds to twice the window size minus one.

Based on the preprocessing, three non-supervised analyses were carried out. The first was based on raw spectra data and called ‘no pretreatment’ while the two
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Moving window PCA algorithm (adapted from by Awhangbo et al. [7])</figcaption></figure>

others used the two most common preprocessing: Standard Normal Variate (SNV, [30]) method and a combination of SNV and the Savitzky-Golay filter (SAVGOL, [31]) which can also be found in the construction of the calibration model made by Ecarnot et al. [28]. The SAVGOL filter was used with a window of 15 nm and a first-degree derivative with a polynomial of the second degree. In total three parameters have varied during the analysis: the window size, the number of components and spectra preprocessing.

In this work, spectra collected at the successive dates from flowering to physiological maturity represented the varying data. MWPCA based approach was proposed here to detect changes that could lead the process to dysfunction and then give an early warning. An abnormal deviation of spectra from those collected before could be considered as an out-of-control state. To define the change in the time-series, we made the hypothesis that Hotelling's T² followed a normal law. The 2.5% higher T² values were considered out of the normal distribution, and representative of a new steady state in the leaf senescence process. Significant changes will not be excluded from the next window contrary to the usual algorithm, because this “new state” of the process will affect the next observations and could even result in a new normal steady state. To compare the results from MWPCA to those from the targeted strategy, the day numbers for which spectra generated significant values of T², named

T² peak, were converted into cumulative GDD. All the analyses were performed with R software [32].

## Results

### Spectra data and leaf senescence process

Typical trans Reflectance spectra of fresh leaves during post the flowering period have been plotted in Fig. 2. The effect of senescence is mainly perceptible in the visible region (around 690 nm), where the continuous degradation of chlorophyll generates a filling of the absorption trough, until it disappears for senescent leaves. The process is also acting in the short wave infrared (SWIR) region (1000–2500 nm) principally shaped by -OH bounds absorption, and mostly water. Indeed, the two most intense water absorption bands are located at 1450 and 1950 nm, and generate troughs in the trans Reflectance spectra, especially for fresh leaves, which then get weaker for senescent leaves. Conversely, the 750–1000 nm spectra region, in which almost no absorption occurs, maintains the same average values all along leaf senescence. Along with the influence of biochemical compounds [33], the physical structure of the leaf determines the average value of the spectra. Leaves with high density have compact mesophylls leading to low scattering, and thus lower average trans Reflectance, which explains the high variance in spectra averages of our data set.
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Raw spectra (from 400 to 2500 nm) of one individual leaf during senescence time course; the darkest blue curves correspond to the first date (flowering) in the experiment while the lightest blue curves indicate the last day of the experiment (end of leaf senescence)</figcaption></figure>

## Supervised models of Nitrogen, Chlorophyll and Water leaf-content prediction

The same pattern, a plateau/drop/plateau sequence, was observed (Fig. 3) for the temporal kinetics of the three leaf biochemical components targeted. Logistic adjustments were highly relevant to report these temporal sequences since the efficiency parameters were close to 1 (>0.95). Based on these curves, two key points, T0 and T1 have been computed for leaf nitrogen, chlorophyll and water content (see Eq. 2). Their average values, as well as their variability for the 139 flag leaves, are reported in Table 1.

Overall, the results show that chlorophyll content was the first to decrease, followed by nitrogen and water contents. The pattern changes for point T1: here the chlorophyll content also ends its variation first, which is followed by water then nitrogen. The standard deviation shows that all the distributions overlap each other, hence the need to treat each individual separately (Fig. 3).

## Unsupervised approach (MWPCA): spectra changes during the senescence process

Based on significant spectra information changes, peak of T² values had been detected for most of the individuals (> 89%). The lowest values were obtained from coupling raw spectra data with a small learning window size (< 5 days) whereas the other combinations provided higher and similar proportions (from 94 to 99%, Table 2).

In addition, on average the number of significant T² values (T² peaks) detected per individual varied from

3 to 4 according to the learning window size and spectra treatment (Fig. 4). This T² peak number was lowest with the raw spectra (about 3.22) and increased according to the pre-treatment process (3.57 and 3.71 for SNV and SNV + Savgol preprocessing respectively). Moreover, if the number of T² peaks observed remained constant whatever the window size with raw spectra information, it decreased as the window size increased for the 2 others spectra preprocessing categories (3.36 < T² < 3.71 and 3.30 < T² < 4.00 for SNV and SNV + Savgol preprocessing respectively).

The information extracted from the MWPCA was condensed in the time interval between onset and end of senescence. To have a better understanding of the information resulting from the two strategies (supervised and unsupervised), we compared the timing given by the T² peaks from MWPCA with T0 and T1 values from supervised modeling which are the signatures of the onset and the end of the leaf senescence process.

Firstly, we linked the earliest peak provided by MWPCA (T0, MWPCA) with the earliest T0 value observed among the three T0 values available (water, chlorophyll or nitrogen) (T0, superv) (Fig. 5). The difference and the correlation between these 2 variables T0, superv and T0, MWPCA were both considered.

Whatever the spectra preprocessing and window size considered, the first peak provided by MWPCA is later than T0, superv, which means that the MWPCA signaled a change in the system after the onset of the senescence (Fig. 5). The range of the differences depended on both
<figure><img src="image_5.png" /><figcaption>**Table 1** Means and standard deviations (n = 139) of T0 and T1 derived from modelling of nitrogen, water and chlorophyll leaf content during the senescence process (accumulated growing GDD post anthesis)</figcaption></figure>

<table><thead><tr><th>Water content T<sub>0</sub></th><th>Nitrogen content T<sub>0</sub></th><th>Chlorophyll content T<sub>0</sub></th><th>Water content T<sub>1</sub></th><th>Nitrogen content T<sub>1</sub></th><th>Chlorophyll content T<sub>1</sub></th></tr></thead><tbody><tr><td>659 ± 100</td><td>530 ± 118</td><td>505 ± 124</td><td>907 ± 129</td><td>916 ± 151</td><td>872 ± 134</td></tr></tbody></table>

pre-treatment and window size: minimum values were obtained with raw spectra and the lowest size window (between -47 and 85 GDD for a window size between 3 and 5). Whatever the spectra pre-treatment, increasing the learning window size amplified these differences (up to 171 GDD).

Furthermore the correlations between T0, MWPCA and T0,Superv were lowest when the differences between the 2 indicators were minimum (raw or SNV spectra coupled with a small learning window size, Fig. 6, r = 0.09). For these two spectra pre-treatments, the correlation values increased according to the size of the window to

<table><caption>**Table 2** Percentage of individuals with at least one significant T² value over the 139 individuals</caption><thead><tr><th rowspan="2">Spectra pre-treatment</th><th colspan="6">Learning window size (number of daily spectra acquisitions)</th></tr><tr><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th></tr></thead><tbody><tr><td>SNV + Savgol</td><td>97.12</td><td>96.40</td><td>97.12</td><td>97.84</td><td>96.40</td><td>94.96</td></tr><tr><td>SNV</td><td>97.84</td><td>97.12</td><td>99.28</td><td>97.84</td><td>99.28</td><td>98.56</td></tr><tr><td>Raw spectra</td><td>89.93</td><td>89.93</td><td>97.84</td><td>93.53</td><td>97.12</td><td>97.84</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>**Fig. 4** Number of T² peaks found in the MWPCA according to the window size expressed in the number of daily spectra included in the reference spectra data set, and the spectra pretreatment</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 5** Difference expressed in GDD between the T0, superv and the T0, MWPCA according to the window size and the pretreatment</figcaption></figure>

reach similar values, whereas for the SNV-Savgol pre-treatment, the correlation remained stable whatever the window size considered. Thus, if similar correlations were obtained in the three pre-treatment with a

large window size, it is noticeable that a more significant spectra pre-treatment provided correlations between T0, MWPCA and T0, superv that were less related to the size of the window.
<figure><img src="image_8.png" /></figure>

Secondly, comparisons between T1, MWPCA and T1, superv led to slightly different observations (Figs. 7 and 8). Unlike with T0, MWPCA provided earlier values for

T1 compared to the supervised model (from 110 to 50 GDD). The spectra pre-treatment affected the range of these differences: it was maximal with raw spectra (~114

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 8** Relationship between T1, superv and T1, MWPCA. The red line corresponds to the 1:1 regression line and the number above each distribution to the correlation between the T1, superv and the T1, MWPCA</figcaption></figure>

GDD) and minimal with the SNV spectra (~50 GDD). Regardless of the pre-treatment, increasing the window size decreased the difference between the two indicators T1, MWPCA and T1, superv.

As for T0 parameters, the use of raw spectra requires higher window size to generate correlation values similar to SNV or SNV-Savgol spectra. When looking at the distribution (Fig. 8), the point clouds show that the T1 from the MWPCA align themselves with the T1 from the supervised model, albeit slightly earlier. Unlike for T0, the point clouds are more constant in their form for all pretreatments and all numbers of components.

## Discussion

Based on fresh leaves, the calibrations obtained using spectral data sets to assess water, nitrogen or chlorophyll content are supported by existing literature [27–29, 34] and provide accurate predictions for the three leaf biochemical variables. Daily measurements on the same flag leaf were used to model the temporal kinetics of the three traits during the senescence process. The shape of the curves was similar for each parameter, and could be described by a sigmoid curve following a pattern ‘plateau/drop/plateau’ with four parameters. The pattern of these temporal dynamics is consistent with that observed for leaf senescence [4, 15].

Based on these curves, two key dates, named T0 and T1 respectively, which are related to the beginning and end of the resorption or desiccation processes, were targeted. Nitrogen fertilization coupled with genetic diversity provided a large variability range for these 2 key points (SD ≥ ±100 °C) whatever the biochemical component similar to results previously reported [35]. On average, T0 mean values of the three-biochemical components varied significantly (mean difference = 154 GDD) whereas T1 values were quite similar (mean difference = 44 GDD).

According to the spectra pre-treatment used, from three to four T² peaks characterizing some leaf spectra changes were detected by the MWPCA. Most of these changes occurred in a time course within the earliest T0, superv (most often related to chlorophyll content) and during the late T1, superv. Window size can sometimes influence the shape of the distribution (Figs. 5B and 6B). The higher number of peaks found by the MWPCA, provides more information, but this information is not always relevant. The choice of the best pretreatment method or window size cannot be based on this parameter alone.

The ability of MWPCA to detect spectra changes according to T0 or T1 computed in the supervised model was uneven. The first peak of the MWPCA was later
than the earliest T0 from the supervised strategy (~100 GDD). Moreover, the correlation between the two data sets remained weak (r < 0.6) indicating that in our study MWPCA based on a global spectra approach is not relevant to signal senescence onset.

MWPCA performed well when used to detect the end of the senescence: the later T² peak was highly correlated with the earlier T1 value obtained using a supervised strategy (up to r=0.86) and the timing of these two key points are similar (~50 GDD). The last peak of the MWPCA seems to correspond to the end of the process, represented by T1.

Senescence duration, i.e. the time between T0 and T1, explains largely variances in the ability of MWPCA to predict T0 and T1. A shorter duration, i.e. a higher senescence rate, induced rapid changes in the biochemical components of leaves, which significantly modified the spectra signature. These bigger changes are better observed by the MWPCA [36] as shown by the strong correlation (up to r=0.80, Fig. 9) between the senescence duration and the discrepancies between the T0, MWPCA and the T0, superv. These data suggest that a non-supervised strategy based on MWPCA as used here will be unable to identify some changes in spectra data if

the properties of the matrix evolve too slowly. This observation is coherent with other studies conducted on others processes [37, 38].

The coupling of no spectra pre-treatment and a small learning window size downgraded the previous results. With these raw spectra data sets, a minimum of a 5-day window size was required to obtain good quality results. We assumed that it could be related to the importance of the variability of overall reflectance amplitude and base line [38]. It has been reported that SNV pretreatment reduces the variability of a spectra data set generated by physical composition heterogeneity of the samples resulting in multiplicative and additive effects [39, 40]. Indeed, differences of micro-environmental conditions from one day to the other could have resulted in heterogeneity in the physical composition of the leaves [41]. Moreover, the position of the leaf in the LeafClip could have been shifted when acquiring the spectrum, so that additive and multiplicative effect could appear. Since the senescence process is more related to biochemical than physical composition, SNV pre-treatment is an appropriate correction to use MWPCA. Reducing the spectral daily unwanted heterogeneity makes it possible to get some results that were less dependent on the window size. The

<figure><img src="image_11.png" /></figure>
minimal window size was reduced to three days instead
of five previously and Savgol pre-treatment allowed us to
obtain similar results whichever the window size.

Looking at the average distance between key points
(T0, T1) computed from supervised and MWPCA meth-
ods, the best pretreatment is SNV, with a window size
from 6 to 8. If the criterion is the correlation coefficient,
the pretreatment and the size of the windows should be
changed according to T0 or T1. SNV pretreatment with
a minimum 5-day window size seems to be the best
compromise.

Prior knowledge of the biological process involved in
senescence is important when choosing some MWPCA
parameters and interpreting results. For example, one
important piece of information to be aware of is that all
the individuals have a plateau (for the first part of their
sigmoidal curve) related to at least eight measurements.
This information can be used to define a priori a range
of window sizes for learning. This was possible because
we had previous knowledge thanks to the supervised
model. This information was important for the MWPCA
because it meant that the learning of the MWPCA could
not be conducted inside the process that was studied.
This knowledge is something that is usually known when
working with the MWPCA.

Conclusions perspectives

Our objective was to manage a complex phenotype
thanks to a non-supervised model applied to whole spec-
tra. Here leaf senescence was considered here as a use
case.

Overall, our study demonstrates that the information
contained in the spectra can be used when applying
an unsupervised method, in this case the MWPCA, to
characterize a complex biological phenomenon such as
leaf span. Of course, further work is needed firstly to
clarify whether the difficulties in predicting some key
points of senescence such as T0 are due to the method
used (i.e. MWPCA) or intrinsic to the unsupervised
strategy and, secondly, to validate this method as a
screening method under field or controlled environ-
ments. For this later point, reducing spectra complex-
ity has to be considered; in relation to light absorbance
in visible (pigments), NIR and SWIR regions (water,
Nitrogen) we observed that reducing the spectra to
the visible or to the SWIR regions (400–1000 nm and
1000–2500 nm, respectively) did not change the results
obtained with the full spectral range (data not shown).
These results suggest that it is possible to downgrade
the device while limiting the risk to document senes-
cence process just on a leaf colour changes. On the
other hand, since some other foliar macronutrients

such as S, Ca, Mg, P and K have a spectral signature
on fresh individual leaves [42], it might be interesting
to document the evolution of their content over the life
span of the wheat leaves, such as done for water, nitro-
gen and chlorophyll. This would allow having a better
understand of the phenomena inducing whole spectral
changes. Finally, to characterise the initial state of the
leaf, spectral pre-processings such as SNV or Savgol
are relevant even with a reduced number of daily spec-
tra included in the reference spectral dataset. Smaller
dataset permits the use of a small training window size,
which is interesting especially when the senescence
process is rapid, such as under conditions of high tem-
perature or mineral depletion.

To conclude, our work suggests that considering the
whole spectra, such as that of a phenotypic trait, based
on a holistic approach, to document complex biological
process may be relevant for tackling both applied issues
in the agricultural context, and forefront research ques-
tions in plant biology.

**Supplementary Information**

The online version contains supplementary material available at https://doi.
org/10.1186/s13007-022-00927-6.

Additional file 1: Table S1. Pretreatments and statistics of the calibration of the supervised model [24].

Acknowledgements

Not applicable.

Author contributions

HV performed analysis on the data. HV, PR, ME wrote the manuscript. EB, ME, PR, RB edited the manuscript. PR supervised the work. All authors have read and approved the final manuscript.

Funding

This work was supported by the French National Research Agency under the Investments for the Future Program, referred as ANR-16-CONV-0004.

Availability of data and materials

The data that support the findings of this study are available from the cor-
responding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have contributed to the research, read the article, and agreed to
submission.

Competing interests

The authors declare that they have no competing interests.

Author details

<footnote n="1">¹AGAP, CIRAD, INRAE, Institut Agro, Univ Montpellier, Montpellier, France.</footnote>

<footnote n="2">²PHIM, CIRAD, INRAE, IRD, Institut Agro, Univ Montpellier, Montpellier, France.</footnote>

<footnote n="3">³ITAP, INRAE, Institut Agro, Univ Montpellier, Montpellier, France.</footnote>
Received: 23 February 2022 Accepted: 18 July 2022

Published online: 12 August 2022

## References

1. Furbank RT, Tester M. Phenomics—technologies to relieve the phenotyp-
ing bottleneck. Trends Plant Sci. 2011;16:635–44.

2. Cobb JN, DeClerck G, Greenberg A, Clark R, McCouch S. Next-generation phenotyping: requirements and strategies for enhancing our understanding of genotype–phenotype relationships and its relevance to crop improvement. Theor Appl Genet. 2013;126:867–87.

3. Watt M, Fiorani F, Usadel B, Rascher U, Muller O, Schurr U. Phenotyp-
ing: new windows into the plant for breeders. Annu Rev Plant Biol.
2020;71:689–712.

4. Anderegg J, Yu K, Aasen H, Walter A, Liebisch F, Hund A. Spectral vegeta-
tion indices to track senescence dynamics in diverse wheat germplasm.
Front Plant Sci. 2020;10:1749.

5. Chapman EA, Orford S, Lage J, Griffiths S. Capturing and selecting senes-
cence variation in wheat. Front Plant Sci. 2021;12: 638738.

6. Kim J, Woo HR, Nam HG. Toward systems understanding of leaf senescence: an integrated multi-omics perspective on leaf senescence research. Mol Plant. 2016;9:813–25.

7. Awhangbo L, Bendoula R, Roger JM, Béline F. Fault detection with mov-
ing window PCA using NIRS spectra for monitoring the anaerobic diges-
tion process. Water Sci Technol. 2020;81:367–82.

8. Schmitt E, Rato T, De Ketelaere B, Reis M, Hubert M. Parameter selection guidelines for adaptive PCA-based control charts: Parameter selection guidelines for adaptive PCA-based control charts. J Chemometrics. 2016;30:163–76.

9. Distelfeld A, Avni R, Fischer AM. Senescence, nutrient remobilization, and yield in wheat and barley. J Exp Bot. 2014;65:3783–98.

10. Camargo Rodriguez AV. Integrative modelling of gene expression and digital phenotypes to describe senescence in wheat. Genes. 2021;12:909.

11. Rouse JW, Haas RH, Deerin DW, Harlan JC. Monitoring the vernal advancement and retrogradation (green wave effect) of natural vegetation. NASA/GSFC Type III Final Report, Greenbelt, Md. 1974. 371.

12. Lim PO, Kim HJ, Gil NH. Leaf senescence. Annu Rev Plant Biol.
2007;58:115–36.

13. Araus JL, Labrana X. Leaf photosynthesis and chloroplast senescence patterns in wheat flag leaves during grain filling. Photosynthetica. 1991;25:33–7.

14. Anderegg J, Aasen H, Perich G, Roth L, Walter A, Hund A. Temporal trends in canopy temperature and greenness are potential indicators of late-season drought avoidance and functional stay-green in wheat. Field Crop Res. 2021;274: 108311.

15. Bogard M, Jourdan M, Allard V, Martre P, Perretant MR, Ravel C, Heumez E, Orford S, Snape J, Griffiths S, Oorbessy G, Foulkes J, Le Gouis J. Anthesis date mainly explained correlations between post-anthesis leaf senescence, grain yield, and grain protein concentration in a winter wheat population segregating for flowering time QTLs. J Exp Bot. 2011;3621:3636–62.

16. Montazeaud G, Karatoğma H, Özturk I, Roumet P, Ecarnot M, Crossa J, et al. Predicting wheat maturity and stay-green parameters by modeling spectral reflectance measurements and their contribution to grain yield under rainfed conditions. Field Crop Res. 2016;196:191–8.

17. Verma V, Foulkes M, Worland A, Sylvester-Bradley R, Caligari P, Snape J. Mapping quantitative trait loci for flag leaf senescence as a yield determinant in winter wheat under optimal and drought-stressed environments. Euphytica. 2004;2004(135):255–63.

18. Christopher JT, Veyradier M, Borrell AK, Harvey G, Fletcher S, Chenu K. Phenotyping novel stay-green traits to capture genetic variation in senescence dynamics. Functional Plant Biol. 2014;41:1035.

19. Thomas H, Howarth CJ. Five ways to stay green. J Exp Bot.
2000;51:329–37.

20. Lopes MS, Saglam D, Ozdogan M, Reynolds M. Traits associated with winter wheat grain yield in Central and West Asia: drought and heat adaptation in winter wheat. J Integr Plant Biol. 2014;56:673–83.

21. Triboi E, Triboi-Blondel A. Productivity and grain or seed composition: a new approach to an old problem. Eur J Agron. 2002;16:163–86.

22. Sultana N, Islam S, Juhasz A, Ma W. Wheat leaf senescence and its regula-
tory gene network. Crop J. 2021;9:703–17.

23. Camargo AV, Mott R, Gardner KA, Mackay IJ, Corke F, Doonan JH, et al.
Determining phenological patterns associated with the onset of senes-
cence in a wheat MAGIC mapping population. Front Plant Sci. 2016.
https://doi.org/10.3389/fpls.2016.01540/full.

24. Gelang J. Rate and duration of grain filling in relation to flag leaf senes-
cence and grain yield in spring wheat (*Triticum aestivum*) exposed to
different concentrations of ozone. Physiol Plant. 2008;110:366–75.

25. Xie Q, Mayes S, Sparkes DL. Early anthesis and delayed but fast leaf senes-
cence contribute to individual grain dry matter and water accumulation
in wheat. Field Crop Res. 2016;187:24–34.

26. Gitelson AA, Merzlyak MN. Spectral reflectance changes associated with autumn senescence of *Aesculus hippocastanum* L. and *Acer platanoides* L. leaves. Spectral features and relation to chlorophyll estimation. J Plant Physiol. 1994;143:286–92.

27. Vilmus I, Ecarnot M, Verzelen N, Roumet P. Monitoring nitrogen leaf resorption kinetics by near-infrared spectroscopy during grain filling in durum wheat in different nitrogen availability conditions. Crop Sci. 2014;54:284–96.

28. Ecarnot M, Compan F, Roumet P. Assessing leaf nitrogen content and leaf mass per unit area of wheat in the field throughout plant cycle with a portable spectrometer. Field Crop Res. 2013;140:44–50.

29. Martens H, Naes T. Multivariate calibration. In: Martens H, Naes T, editors. J Chemometrics. New York: Wiley; 1989.

30. Barnes RJ, Dhanoa MS, Lister SJ. Standard normal variate transformation and de-trending of near-infrared diffuse reflectance spectra. Appl Spectrosc. 1989;43:772–7.

31. Savitzky A, Golay MJE. Smoothing and differentiation of data by simpli-
fied least squares procedures. Anal Chem. 1964;36:1627–39.

32. R Development Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria; 2012. http://www.r-project.org/

33. Gausman HW. Leaf Reflectance of Near-infrared. Photogramm Eng. 1974:40:183–91.

34. Wang S, Guan K, Wang Z, Ainsworth EA, Zheng T, Townsend PA, et al. Unique contributions of chlorophyll and nitrogen to predict crop photo-synthetic capacity from leaf spectroscopy. J Exp Bot. 2021;72:341–54.

35. Hafsi M, Mechmeche W, Bouamama L, Djekoue A, Zaharieva M, Monneveux P. Flag leaf senescence, as evaluated by numerical image analysis, and its relationship with yield under drought in durum wheat. J Agron Crop Sci. 2000;185:275–80.

36. Kruger U, Xie L. Statistical monitoring of complex multivariate processes with applications in industrial process control. Chichester: John Wiley & Sons Ltd; 2012.

37. Ayech N, Chakour C, Harkat M-F. New adaptive moving window PCA for process monitoring. IFAC Proceedings Volumes. 2012;45:606–11.

38. Haimi H, Mulas M, Corona F, Marsili-Libelli S, Lindell P, Heinonen M, et al. Adaptive data-derived anomaly detection in the activated sludge process of a large-scale wastewater treatment plant. Eng Appl Artif Intell. 2016;52:65–80.

39. Lehmann J, Große-Stoltenberg A, Römer M, Oldeland J. Field spectroscopy in the VNIR-SWIR region to discriminate between mediterranean native plants and exotic-invasive shrubs based on leaf tannin content. Remote Sens. 2015;7:1225–41.

40. Hellmann C, Große-Stoltenberg A, Lauströ V, Oldeland J, Werner C. Retrieving nitrogen isotopic signatures from fresh leaf reflectance spectra: disentangling δ15N from biochemical and structural leaf properties. Front Plant Sci. 2015. https://doi.org/10.3389/fpls.2015.00307.

41. Chatterton NJ, Lee DR, Hungerford WE. Diurnal change in specific leaf weight of *Medicago sativa* and *Zea mays*. Crop Sci. 1972;12:576–8.

42. Prananto JA, Minasny B, Weaver T. Rapid and cost-effective nutrient content analysis of cotton leaves using near-infrared spectroscopy (NIRS). Peer J. 2021;9:e11042.

## Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.