Characterization of plant water flows in Controlled environment -PLANT SMART SENSORS

The present thesis project "Characterization of water flows in Controlled Environment -PLANT SMART SENSORS" has a multidisciplinary core and aimed towards the creation of synergies between the world of scientific research and the industry. By applying research results to technological development, this research targeted at innovation in the Agrotechnology and Aerospace sectors. Indeed, the introduction of new technologies is pivotal for controlled environment production on Earth to feed a growing population as well as for human permanence in Space in longterm missions where plants are used to regenerate resources (e.g. oxygen, water) and as source of fresh high-nutritious food. The realization of these systems must be based on a precise knowledge of plant morpho-anatomical development and its physiological behavior in closed growth systems, which are strongly influenced by numerous environmental factors including the relative humidity or more specifically the Vapour Pressure Deficit (VPD). In a protected environment (e.g. in Space greenhouses, vertical farm, indoor growing-modules), the control of relative humidity represents a significant problem, which has often been neglected. For instance, in conditions of poor aeration, too high humidity can occur with consequent low values of VPD which reduces the plant transpiration, slowing or stopping the water flow through the SPAC (Soil-plant-atmosphere-continuum), and ultimately blocking the photosynthesis, yield and biomass production. Even though there have been many studies regarding the VPD control, alone and/or in combination with other environmental factors, certain points are still unclear or controversial, providing contrasting results in different or even in the same species. This happens mainly due to the complex interactions between many microclimatic factors and plant physiological behaviour at different phenological stages. In a context of climate change, the efficient regulation of VPD can be applied to greenhouse and indoor-module production in order to enhance crop productivity, improve WUE and reduce total water consumption to design irrigation strategies, considering the balance between the amount of water saved and the quantity used to regulate the VPD. The regulation of the VPD and related environmental parameters need to be designed according to the species and its adaptive plasticity at morphophysiological levels. Thus, the characterization and modeling of water flows in model plants in different growth chamber scenarios (from small modules intended for the spatialization for Space applications, up to structures that can be used in protected cultivation on Earth), as well as the real-time monitoring of the water status of plants, become fundamental for the management of precision agriculture both in support of Space exploration and for the sustainability of urban agriculture. To date, most of the research has focused on either specific physiological/structural aspect at the single-plant level, or on cultivation management or even on technological aspects, with only a few interlinks of knowledge. The aim of this thesis is to develop knowledge to help filling this gap to improve the understanding of VPD effects on crop productivity, with the creation of synergies among different expertise (e.g., plant physiology, crop science, engineering). To do so, it is fundamental to study the complexity of plant morpho/physiological responses, since without a deep knowledge of mechanisms behind plant responses to the environment it is difficult to determine how and to which extent plants can adapt to any changes in the environmental conditions. The application of a multidisciplinary approach in research will allow crop production in a sustainable way, even in harsh environments, where a "climate smart-agriculture" becomes necessary to improve crop yield and quality. The present thesis is organized as follows: Chapter 1 is a review which presents the current state of knowledge on how VPD influences plant morpho-physiological traits in controlled environment agriculture. The study has been published as a review article in Annals of Applied Biology (Amitrano et al., 2019 https://doi.org/10.1111/aab.12544). It covers main important aspects of VPD influence on plant growth, morpho-anatomical development, and physiology, emphasizing the possible interaction between VPD and other microclimatic factors in protected cultivation. Furthermore, the rewiew identifies and discusses future research areas, which should be explored further, based on needed synergies among different expertise from biological and horticultural fields. Chapter 2 presents evidence that the modulation of relative humidity (RH) together with other important cultivation factors such as light (presence/absence), can influence morpho-anatomical development and improve antioxidant content, even at the early stages of plant life cycle (germination, seedling establishment). The combined effect of RH and light was studied during the germination and seedling development of Vigna radiata L. (mung bean), a species widespread throughout the world also due to the high nutritional value of its edible sprouts. A manuscript reporting these data has been published in Plants (Amitrano et al., 2020a https://doi.org/10.3390/plants9091093). In Chapter 3, the role of leaf anatomical traits (e.g. leaf mesophyll features, stomata and vein traits) in photosynthetic acclimation to short- and long-term changes in VPD was examined in Vigna radiata L. adult plants. In this study, we underlined the key role of leaf structure in photosynthetic acclimation to air VPD. The long-term exposure to different VPD levels determined a pre-acclimation at the leaf morpho-anatomical level which influenced the extent of leaf physiological plasticity, changing plant ability to acclimate to any changes in the surrounding microclimate. This different leaf anatomy-related capacity of pre-acclimating becomes therefore fundamental in the present climate-change scenario due to its key role in the adaptation process under changing environmental conditions. A manuscript reporting these data has been published in Environmental and Experimental Botany (Amitrano et al., 2021a https://doi.org/10.1016/j.envexpbot.2021.104453). In Chapter 4, the effect of VPD on morpho-physiological traits also incorporating the trade-off between transpiration and carbon gain was evaluated in two cultivars of Salanova lettuce (Lactuca sativa L.) with green and red leaves, in a growth-chamber experiment. Low-VPD turned out to significantly improve growth, stomata development and hydraulic-related traits which led to higher photosynthesis and a reduced water consumption compared to the high-VPD condition. A manuscript reporting these data was published in Agronomy (Amitrano et al., 2021b https://doi.org/10.3390/agronomy11071396). Chapter 5 represents a clear interlink of knowledge between plant scientists, engineers, mathematician and modelists. In this study, published in Sensors (Amitrano et al., 2020b https://doi.org/10.3390/s20113110), we used experimental data, based on morpho-anatomical analyses of lettuce plants, to run the Energy Cascade Model (MEC), a model already used to predict biomass production and photosynthetic efficiency in advanced life support systems studies (Space-oriented research). Here, the modification of the model is discussed together with possible improvements and applications. Chapter 6 focuses on how to modulate the micro-environment, and in particular the VPD levels, in protected cultivation to improve plant antioxidant content in crops. More specifically, the exposure of the same lettuce cultivars mentioned in previous chapters to high VPD determined an improved phytochemical content in lettuce leaves, especially in the red cultivar. Here we discussed a further possibility to use short-term high VPD treatments as a mild stress to boost the phytochemical production in lettuce plants. A Manuscript reporting these data has been published in Horticulturae (Amitrano et al., 2021c http://doi.org/10.3390/horticulturae7020032). Chapter 7 is a deep focus on how the VPD drives the coordination among morpho-anatomical traits in leaves of the above-mentioned lettuce cultivars, also exploring the variability of traits along the leaf lamina. More specifically, the attention is focused on how stomata and vein develop within lettuce leaves and how these traits are coordinated with leaf size under different VPDs. Results from this study suggest that VPD triggers a different response in lettuce plants in terms of balance of leaf 4 traits and highlight the possibility of further exploring the microenvironment (combined influence of light and VPD) to adjust the development of stomata and vein densities, thus providing optimal water and gas fluxes through the leaves. In Chapter 8, the experiments conducted during the period spent at the Controlled Environment Agriculture Center of the University of Arizona (UA-CEAC) are reported. The experiments reported here were conducted on the same species of the previous chapters (Salanvoa lettuce with green and red leaves) in a multi-layer vertical farm to test the interaction between VPD and other microclimatic factors on plant morpho-physiological development. More specifically two experimental trials are reported (E1 and E2). In E1, the interaction between VPD levels (low and high) and increasing DLI (Daily Light Integral - 8.6, 12.9, 15.5) was tested to study morpho-physiological changes and to determine the optimal combination of DLI and VPD for lettuce growth. In E2, a sudden salt stress was applied to the cultivation and then CO2 enrichment was provided, based on the hypothesis that the CO2 enrichment would mitigate the salt stress, modifying the plant carbon gain/water balance. We evaluated whether the mechanisms of salt stress mitigation due to CO2 enrichment were different under high and low VPD conditions, depending on the different morphoanatomical leaf structure. Chapter 9 reports on experiments conducted at the IPK-Leibniz institute of plant genetics and crop plant research (Gatersleeben, Germany) in the framework of the EPPN2020 transnational access (https://eppn2020.plant-phenotyping.eu/EPPN_Transnational_Access). A report with obtained results is showed in this chapter. These experiments concern the application of high-throughput phenotyping combined with morpho-anatomical analyses on Salanova green and red plants acclimated to a VPD level and then subjected to short-term changes in the VPD. The project submitted to the EPPN transnational access and winner of the grant is presented in Appendix 1. Chapter 10 and 11 report on the possible industrial applications after the collaboration with the partner company "Kayser Italia srl" (http://www.kayser.it/). Chapter 10 is a study for the definition of scientific and technical requirements for the realization of a miniaturized phenotyping growth chamber to grow microgreens or small crops in Space. The structure of the chamber is based on the "Kubik" incubator, an incubator facility of the European Space Agency with the shape of a cube of about 40 cm that has been operating aboard the International Space Station for more than 12 years, carrying different life science experiments. In the chapter, technical and scientific requirements are listed and a preliminary schedule for the project realization is provided. At the end of the chapter, open issues are also discussed. In Chapter 11, the set-up of a prototype miniaturized cultivation chamber for use in Space is described and the results of validation tests, carried out at Kayser Italia with brassica microgreens (Brassica rapa subsp. sylvestris var. esculenta) under different air relative humidities (VPD), are reported. In Appendix 1, the project submitted to the EPPN transnational access (PHEW- Automated phenotyping platform to improve lettuce water use efficiency under different VPD and watering regimens) and winner of the grant is presented. In Appendix 2, a brief recap on the activities conducted during the Ph.D. program is presented