Water supply limitations will likely impose increasing restrictions on future crop production, underlining a need for crops that use less water per mass of yield. Water use efficiency (WUE) therefore becomes a key consideration in developing resilient and productive crops. In this study, we hypothesized that it is possible to improve WUE under drought conditions via modulation of chloroplast signals for stomatal opening by up-regulation of non-photochemical quenching (NPQ). Nicotiana tabacum plants with strong overexpression of the PsbS gene encoding PHOTOSYSTEM II SUBUNIT S, a key protein in NPQ, were grown under differing levels of drought. The PsbS-overexpressing lines lost 11% less water per unit CO₂ fixed under drought and this did not have a significant effect on plant size. Depending on growth conditions, the PsbS-overexpressing lines consumed from 4–30% less water at the whole-plant level than the corresponding wild type. Leaf water and chlorophyll contents showed a positive relation with the level of NPQ. This study therefore provides proof of concept that up-regulation of NPQ can increase WUE, and as such is an important step towards future engineering of crops with improved performance under drought.

Incorporating cover crops into cropping systems offers numerous potential benefits, including the reduction of soil erosion, suppression of weeds, decreased nitrogen requirements for subsequent crops, and increased carbon sequestration. The aboveground biomass (AGB) of cover crops strongly influences their performance in delivering these benefits. Despite the significance of AGB, a comprehensive field-based high-throughput phenotyping study to quantify AGB of multiple cover crops in the U.S. Midwest has not been found. This study presents a two-year field experiment carried out in Eastern Nebraska, USA, to estimate AGB of five different cover crop species [canola (Brassica napus L.), rye (Secale cereale L.), triticale (Triticale × Triticosecale L.), vetch (Vicia sativa L.), and wheat (Triticum aestivum L.)] using high-throughput phenotyping and Machine Learning (ML) models. Destructive AGB sampling was performed three times during each spring season in 2022 and 2023. An array of morphological, spectral, thermal, and environmental features from the sensors were utilized as feature inputs of ML models. Moderately strong linear correlations between AGB and the selected features were observed. Four ML models, namely Random Forests Regression (RFR), Support Vector Regression (SVR), Partial Least Squares Regression (PLSR), and Artificial Neural Network (ANN), were investigated. Among the four models, PLSR achieved the highest Coefficient of Determination (R²) of 0.84 and the lowest Root Mean Squared Error (RMSE) of 892 kg/ha (Normalized RMSE (NRMSE) = 8.87%), indicating that PLSR could be the most appropriate method for estimating AGB of multiple cover crop species. Feature importance analysis ranked spectral features like Normalized Difference Red Edge (NDRE), Solar-induced Fluorescence (SIF), Spectral Reflectance at 485 nm (R485), and Normalized Difference Vegetation Index (NDVI) as top model features using PLSR. When utilizing fewer feature inputs, ANN exhibited better prediction performance compared to other models. Using morphological and spectral parameters as input features alone led to a R² of 0.80 and 0.77 for AGB prediction using ANN, respectively. This study demonstrated the feasibility of high-throughput phenotyping and ML techniques for accurately estimating AGB of multiple cover crop species. Further enhancement of model performance could be achieved through additional destructive sampling conducted across multiple locations and years.

Advancements in imaging, computer vision, and automation have revolutionized various fields, including field-based high-throughput plant phenotyping (FHTPP). This integration allows for the rapid and accurate measurement of plant traits. Deep Convolutional Neural Networks (DCNNs) have emerged as a powerful tool in FHTPP, particularly in crop segmentation—identifying crops from the background—crucial for trait analysis. However, the effectiveness of DCNNs often hinges on the availability of large, labeled datasets, which poses a challenge due to the high cost of labeling. In this study, a deep learning with bagging approach is introduced to enhance crop segmentation using high-resolution RGB images, tested on the NU-Spidercam dataset from maize plots. The proposed method outperforms traditional machine learning and deep learning models in prediction accuracy and speed. Remarkably, it achieves up to $40\%$ higher Intersection-over-Union (IoU) than the threshold method and $11\%$ over conventional machine learning, with significantly faster prediction times and manageable training duration. Crucially, it demonstrates that even small labeled datasets can yield high accuracy in semantic segmentation. This approach not only proves effective for FHTPP but also suggests potential for broader application in remote sensing, offering a scalable solution to semantic segmentation challenges. This paper is accompanied by publicly available source code.

Nitrogen Sufficiency Index (NSI) is an important nitrogen (N) stress indicator for precision N management. It is usually calculated using variables such as leaf chlorophyll meter readings (SPAD) and vegetation indices (VIs). However, no consensus has been reached on the most preferred variable. Additionally, conventional NSI (NSI_uni) calculation assumes N being the sole yield-limiting factor, neglecting other factors such as soil water variability. To tackle these issues, this study compared various variables for NSI calculation and evaluated two new N stress indicators in minimizing the impact of confounding water treatment. The following ground- and aerial-derived variables were compared for NSI_uni calculation: SPAD, sampled leaf and canopy N content (LNC, CNC), LNC and CNC estimated using hyperspectral images acquired by an Unmanned Aerial Vehicle, and three VIs (Normalized Difference Vegetation Index (NDVI), Normalized Red Edge Index (NDRE), and Chlorophyll Index) from the hyperspectral images. Results demonstrated that ground-measured variables outperformed aerial-based variables in deriving N-responsive NSI. Especially, LNC derived NSI_uni responded to N treatment significantly in ten out of thirteen site-date datasets. For the second objective, a modified NSI (NSI_w) and the NDRE/NDVI ratio were compared to NSI_uni. NSI_w reduced water treatment effects in over 80% of the datasets where NSI_uni showed evident impacts. NDRE/NDVI performed similarly to NSI_w, with the notable advantage of not requiring prior knowledge of soil water spatial distribution. This research pioneers the optimization of N stress indicators by identifying the best variables for NSI and mitigating the effects of soil water variability. These advancements significantly contribute to precision N management in complex field conditions.