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Gravity is a powerful element in shaping plant development, with gravitropism, the oriented growth response of plant organs to the direction of gravity, leading to each plant’s characteristic form both above and below ground. Despite being conceptually simple to follow, monitoring a plant’s directional growth responses can become complex as variation arises from both internal developmental cues as well as effects of the environment. In this protocol, we discuss approaches to gravitropism assays, focusing on automated analyses of root responses. For Arabidopsis, we recommend a simple 90􏰁 rotation using seedlings that are 5–8 days old. If images are taken at regular intervals and the environmental metadata is recorded during both seedling development and gravitropic assay, these data can be used to reveal quantitative kinetic patterns at distinct stages of the assay. The use of software that analyzes root system parameters and stores this data in the RSML format opens up the possibility for a host of root parameters to be extracted to characterize growth of the primary root and a range of lateral root phenotypes. 

Abstract A plant’s oxygen supply can vary from normal (normoxia) to total depletion (anoxia). Tolerance to anoxia is relevant to wetland species, rice (Oryza sativa) cultivation, and submergence tolerance of crops. Decoding and transmitting calcium (Ca) signals may be an important component to anoxia tolerance; however, the contribution of intracellular Ca transporters to this process is poorly understood. Four functional cation/proton exchangers (CAX1–4) in Arabidopsis (Arabidopsis thaliana) help regulate Ca homeostasis around the vacuole. Our results demonstrate that cax1 mutants are more tolerant to both anoxic conditions and submergence. Using phenotypic measurements, RNA-sequencing, and proteomic approaches, we identified cax1-mediated anoxia changes that phenocopy changes present in anoxia-tolerant crops: altered metabolic processes, diminished reactive oxygen species production post anoxia, and altered hormone signaling. Comparing wild-type and cax1 expressing genetically encoded Ca indicators demonstrated altered cytosolic Ca signals in cax1 during reoxygenation. Anoxia-induced Ca signals around the plant vacuole are involved in the control of numerous signaling events related to adaptation to low oxygen stress. This work suggests that cax1 anoxia response pathway could be engineered to circumvent the adverse effects of flooding that impair production agriculture.