Automated Imaging, Tracking, and Analytics Pipeline for Differentiating Environmental Effects on Root Meristematic Cell Division

Abstract

Exposure of plants to abiotic stresses, whether individually or in combination, triggers dynamic changes to gene regulation. These responses induce distinct changes in phenotypic characteristics, enabling the plant to adapt to changing environments. For example, iron deficiency and heat stress have been shown to alter root development by reducing primary root growth and reducing cell proliferation, respectively. Currently, identifying the dynamic temporal coordination of genetic responses to combined abiotic stresses remains a bottleneck. This is, in part, due to an inability to isolate specific intervals in developmental time where differential activity in plant stress responses plays a critical role. Here, we observed that iron deficiency, in combination with temporary heat stress, suppresses the expression of iron deficiency-responsive pPYE::LUC (POPEYE::luciferase) and pBTS::LUC (BRUTUS::luciferase) reporter genes. Moreover, root growth was suppressed less under combined iron deficiency and heat stress than under either single stress condition. To further explore the interaction between pathways, we also created a computer vision pipeline to extract, analyze, and compare high-dimensional dynamic spatial and temporal cellular data in response to heat and iron deficiency stress conditions at high temporal resolution. Specifically, we used fluorescence light sheet microscopy to image Arabidopsis thaliana roots expressing CYCB1;1:GFP, a marker for cell entry into mitosis, every 20 min for 24 h exposed to either iron sufficiency, iron deficiency, heat stress, or combined iron deficiency and heat stress. Our pipeline extracted spatiotemporal metrics from these time-course data. These metrics showed that the persistency and timing of CYCB1;1:GFP signal were uniquely different under combined iron deficiency and heat stress conditions versus the single stress conditions. These metrics also indicated that the spatiotemporal characteristics of the CYCB1;1:GFP signal under combined stress were more dissimilar to the control response than the response seen under iron deficiency for the majority of the 24-h experiment. Moreover, the combined stress response was less dissimilar to the control than the response seen under heat stress. This indicated that pathways activated when the plant is exposed to both iron deficiency and heat stress affected CYCB1;1:GFP spatiotemporal function antagonistically

Ferroptosis in plants: triggers, proposed mechanisms, and the role of iron in modulating cell death

“…Heat stress is also able to induce ferroptosis in a very specific manner (Distéfano et al, 2017). Even when there is not a master regulator described for stress response in plants, several metabolic and transcriptional changes are commonly seen in response to different types of environmental stimuli (Buckner et al, 2019). Ferroptosis, in fact, involves molecular events such as ROS accumulation, activation of lipid peroxidation and antioxidant depletion that have been described after several kinds of insults (Senda and Ogawa, 2004;Pavet et al, 2005;Van Breusegem and Dat, 2006;Doyle et al, 2010;Noctor et al, 2012;Xie et al, 2014).…”

“…These transcription factors are induced in response to iron deficiency (García et al, 2018;Rodríguez-Celma et al, 2019). Interestingly, iron deficiency in combination with heat stress downregulates the expression of iron deficiency-responsive genes, which might ameliorate heat stress-induced ferroptosis in plants (Buckner et al, 2019). Similarly, silencing of IREB2, which encodes a protein that senses iron levels and controls the expression of many other proteins related to iron transport, storage and turnover, attenuates erastin-induced ferroptosis in cancer cells (Dixon et al, 2012).…”

Establishing a reproducible approach for the controllable deposition and maintenance of plants cells with 3D bioprinting

“…The following lines were used: wild type, pEN7:GFP (AT4G28100) (33), pSCR:SCR-mCherry (AT3G54220) (34), and pCYCB1;1:CYCB1;1-GFP (AT4G37490) (35) in Columbia-0 background, and J0571 (30) in the C24 background. Arabidopsis thaliana seeds were surface sterilized using 50% bleach and 0.05% Tween-20 for 5 minutes, followed by a 2 minute incubation in 70% ethanol for 2 minutes.…”

Imaging plant cells and organs with light-sheet and super-resolution microscopy

“…In most recent applications, LSFM is still very popular and efficient in the elucidation of Arabidopsis root development. It was used in the analysis of root growth dynamics and cell cycle patterns over time under iron deficiency and in combination with temporary heat stress by tracking of CYCLINB1;1-GFP (CYCB1;1-GFP), a marker for cell entry into mitosis ( Buckner et al, 2019 ). Multi-dimensional time-course LSFM study and cell file tracking in Arabidopsis root reacting to different water availability, showed the flexibility of lateral root development during the root system branching.…”