Development of a mobile, high-throughput, and low-cost image-based plant growth phenotyping system.

Nondestructive plant phenotyping forms a key technique for unraveling molecular processes underlying plant development and response to the environment. While the emergence of high-throughput phenotyping facilities can further our understanding of plant development and stress responses, their high costs greatly hinder scientific progress. To democratize high-throughput plant phenotyping, we developed sets of low-cost image- and weight-based devices to monitor plant shoot growth and evapotranspiration. We paired these devices to a suite of computational pipelines for integrated and straightforward data analysis. The developed tools were validated for their suitability for large genetic screens by evaluating a cowpea (Vigna unguiculata) diversity panel for responses to drought stress. The observed natural variation was used as an input for a genome-wide association study, from which we identified nine genetic loci that might contribute to cowpea drought resilience during early vegetative development. The homologs of the candidate genes were identified in Arabidopsis (Arabidopsis thaliana) and subsequently evaluated for their involvement in drought stress by using available T-DNA insertion mutant lines. These results demonstrate the varied applicability of this low-cost phenotyping system. In the future, we foresee these setups facilitating the identification of genetic components of growth, plant architecture, and stress tolerance across a wide variety of plant species.

Arabinosylation of cell wall extensin is required for the directional response to salinity in roots.

Soil salinity is a major contributor to crop yield losses. To improve our understanding of root responses to salinity, we developed and exploited a real-time salt-induced tilting assay. This assay follows root growth upon both gravitropic and salt challenges, revealing that root bending upon tilting is modulated by Na+ ions, but not by osmotic stress. Next, we measured this salt-specific response in 345 natural Arabidopsis (Arabidopsis thaliana) accessions and discovered a genetic locus, encoding the cell wall-modifying enzyme EXTENSIN ARABINOSE DEFICIENT TRANSFERASE (ExAD) that is associated with root bending in the presence of NaCl (hereafter salt). Extensins are a class of structural cell wall glycoproteins known as hydroxyproline (Hyp)-rich glycoproteins, which are posttranslationally modified by O-glycosylation, mostly involving Hyp-arabinosylation. We show that salt-induced ExAD-dependent Hyp-arabinosylation influences root bending responses and cell wall thickness. Roots of exad1 mutant seedlings, which lack Hyp-arabinosylation of extensin, displayed increased thickness of root epidermal cell walls and greater cell wall porosity. They also showed altered gravitropic root bending in salt conditions and a reduced salt-avoidance response. Our results suggest that extensin modification via Hyp-arabinosylation is a unique salt-specific cellular process required for the directional response of roots exposed to salinity.

The nuclear lamina is required for proper development and nuclear shape distortion in tomato.

The nuclear lamina in plant cells is composed of plant-specific proteins, including nuclear matrix constituent proteins (NMCPs), which have been postulated to be functional analogs of lamin proteins that provide structural integrity to the organelle and help stabilize the three-dimensional organization of the genome. Using genomic editing, we generated alleles for the three genes encoding NMCPs in cultivated tomato (Solanum lycopersicum) to determine if the consequences of perturbing the nuclear lamina in this crop species were similar to or distinct from those observed in the model Arabidopsis thaliana. Loss of the sole NMCP2-class protein was lethal in tomato but is tolerated in Arabidopsis. Moreover, depletion of NMCP1-type nuclear lamina proteins leads to distinct developmental phenotypes in tomato, including leaf morphology defects and reduced root growth rate (in nmcp1b mutants), compared with cognate mutants in Arabidopsis. These findings suggest that the nuclear lamina interfaces with different developmental and signaling pathways in tomato compared with Arabidopsis. At the subcellular level, however, tomato nmcp mutants resembled their Arabidopsis counterparts in displaying smaller and more spherical nuclei in differentiated cells. This result argues that the plant nuclear lamina facilitates nuclear shape distortion in response to forces exerted on the organelle within the cell.

NIGT1.4 maintains primary root elongation in response to salt stress through induction of ERF1 in Arabidopsis.

Plants employ various molecular mechanisms to maintain primary root elongation upon salt stress. Identification of key functional genes, therein, is important for improving crop salt tolerance. Through analyzing natural variation of the primary root length of Arabidopsis natural population under salt stress, we identified NIGT1.4, encoding an MYB transcription factor, as a novel contributor to maintained root growth under salt stress. Using both T-DNA knockout and functional complementation, NIGT1.4 was confirmed to have a role in promoting primary root growth in response to salt stress. The expression of NIGT1.4 in the root was shown induced by NaCl treatments in an ABA-dependent manner. SnRK2.2 and 2.3 were shown to interact with and phosphorylate NIGT1.4 individually. The growth of the primary root of snrk2.2/2.3/2.6 triple mutant was shown sensitive to salt stress, which was similar to nigt1.4 plants. Using DNA affinity purification sequencing, ERF1, a known positive regulator for primary root elongation and salt tolerance, was identified as a target gene for NIGT1.4. The transcriptional induction of ERF1 by salt stress was shown absent in nigt1.4 background. NIGT1.4 was also confirmed to bind to the promoter region of ERF1 by yeast one-hybrid experiment and to induce the expression of ERF1 by dual-luciferase analysis. All data support the notion that salt- and ABA-elicited NIGT1.4 induces the expression of ERF1 to regulate downstream functional genes that contribute to maintained primary root elongation. NIGT1.4-ERF1, therefore, acts as a signaling node linking regulators for stress resilience and root growth, providing new insights for breeding salt-tolerant crops.

Tapping into the plasticity of plant architecture for increased stress resilience.

Plant architecture develops post-embryonically and emerges from a dialogue between the developmental signals and environmental cues. Length and branching of the vegetative and reproductive tissues were the focus of improvement of plant performance from the early days of plant breeding. Current breeding priorities are changing, as we need to prioritize plant productivity under increasingly challenging environmental conditions. While it has been widely recognized that plant architecture changes in response to the environment, its contribution to plant productivity in the changing climate remains to be fully explored. This review will summarize prior discoveries of genetic control of plant architecture traits and their effect on plant performance under environmental stress. We review new tools in phenotyping that will guide future discoveries of genes contributing to plant architecture, its plasticity, and its contributions to stress resilience. Subsequently, we provide a perspective into how integrating the study of new species, modern phenotyping techniques, and modeling can lead to discovering new genetic targets underlying the plasticity of plant architecture and stress resilience. Altogether, this review provides a new perspective on the plasticity of plant architecture and how it can be harnessed for increased performance under environmental stress.

A Mathematical Framework for Analyzing Wild Tomato Root Architecture.

The root architecture of wild tomato, Solanum pimpinellifolium, can be viewed as a network connecting the main root to various lateral roots. Several constraints have been proposed on the structure of such biological networks, including minimizing the total amount of wire necessary for constructing the root architecture (wiring cost), and minimizing the distances (and by extension, resource transport time) between the base of the main root and the lateral roots (conduction delay). For a given set of lateral root tip locations, these two objectives compete with each other-optimizing one results in poorer performance on the other-raising the question how well S. pimpinellifolium root architectures balance this network design trade-off in a distributed manner. In this study, we describe how well S. pimpinellifolium roots resolve this trade-off using the theory of Pareto optimality. We describe a mathematical model for characterizing the network structure and design trade-offs governing the structure of S. pimpinellifolium root architecture. We demonstrate that S. pimpinellifolium arbors construct architectures that are more optimal than would be expected by chance. Finally, we use this framework to quantify structural differences between arbors grown in the presence of salt stress, classify arbors into four distinct architectural ideotypes, and test for heritability of variation in root architecture structure.

Genomic impact of stress-induced transposable element mobility in Arabidopsis.

Transposable elements (TEs) have long been known to be major contributors to plant evolution, adaptation and crop domestication. Stress-induced TE mobilization is of particular interest because it may result in novel gene regulatory pathways responding to stresses and thereby contribute to stress adaptation. Here, we investigated the genomic impacts of stress induced TE mobilization in wild type Arabidopsis plants. We find that the heat-stress responsive ONSEN TE displays an insertion site preference that is associated with specific chromatin states, especially those rich in H2A.Z histone variant and H3K27me3 histone mark. In order to better understand how novel ONSEN insertions affect the plant's response to heat stress, we carried out an in-depth transcriptomic analysis. We find that in addition to simple gene knockouts, ONSEN can produce a plethora of gene expression changes such as: constitutive activation of gene expression, alternative splicing, acquisition of heat-responsiveness, exonisation and genesis of novel non-coding and antisense RNAs. This report shows how the mobilization of a single TE-family can lead to a rapid rise of its copy number increasing the host's genome size and contribute to a broad range of transcriptomic novelty on which natural selection can then act.

A community-led initiative for training in reproducible research.

Open and reproducible research practices increase the reusability and impact of scientific research. The reproducibility of research results is influenced by many factors, most of which can be addressed by improved education and training. Here we describe how workshops developed by the Reproducibility for Everyone (R4E) initiative can be customized to provide researchers at all career stages and across most disciplines with education and training in reproducible research practices. The R4E initiative, which is led by volunteers, has reached more than 3000 researchers worldwide to date, and all workshop materials, including accompanying resources, are available under a CC-BY 4.0 license at https://www.repro4everyone.org/.

Genetic mapping of the early responses to salt stress in Arabidopsis thaliana.

Salt stress decreases plant growth prior to significant ion accumulation in the shoot. However, the processes underlying this rapid reduction in growth are still unknown. To understand the changes in salt stress responses through time and at multiple physiological levels, examining different plant processes within a single set-up is required. Recent advances in phenotyping has allowed the image-based estimation of plant growth, morphology, colour and photosynthetic activity. In this study, we examined the salt stress-induced responses of 191 Arabidopsis accessions from 1 h to 7 days after treatment using high-throughput phenotyping. Multivariate analyses and machine learning algorithms identified that quantum yield measured in the light-adapted state (Fv' /Fm' ) greatly affected growth maintenance in the early phase of salt stress, whereas the maximum quantum yield (QYmax ) was crucial at a later stage. In addition, our genome-wide association study (GWAS) identified 770 loci that were specific to salt stress, in which two loci associated with QYmax and Fv' /Fm' were selected for validation using T-DNA insertion lines. We characterized an unknown protein kinase found in the QYmax locus that reduced photosynthetic efficiency and growth maintenance under salt stress. Understanding the molecular context of the candidate genes identified will provide valuable insights into the early plant responses to salt stress. Furthermore, our work incorporates high-throughput phenotyping, multivariate analyses and GWAS, uncovering details of temporal stress responses and identifying associations across different traits and time points, which are likely to constitute the genetic components of salinity tolerance.

The role of PQL genes in response to salinity tolerance in Arabidopsis and barley.

While soil salinity is a global problem, how salt enters plant root cells from the soil solution remains underexplored. Non-selective cation channels (NSCCs) are suggested to be the major pathway for the entry of sodium ions (Na+), yet their genetic constituents remain unknown. Yeast PQ loop (PQL) proteins were previously proposed to encode NSCCs, but the role of PQLs in plants is unknown. The hypothesis tested in this research is that PQL proteins constitute NSCCs mediating some of the Na+ influx into the root, contributing to ion accumulation and the inhibition of growth in saline conditions. We identified plant PQL homologues, and studied the role of one clade of PQL genes in Arabidopsis and barley. Using heterologous expression of AtPQL1a and HvPQL1 in HEK293 cells allowed us to resolve sizable inwardly directed currents permeable to monovalent cations such as Na+, K+, or Li+ upon membrane hyperpolarization. We observed that GFP-tagged PQL proteins localized to intracellular membrane structures, both when transiently over-expressed in tobacco leaf epidermis and in stable Arabidopsis transformants. Expression of AtPQL1a, AtPQL1b, and AtPQL1c was increased by salt stress in the shoot tissue compared to non-stressed plants. Mutant lines with altered expression of AtPQL1a, AtPQL1b, and AtPQL1c developed larger rosettes in saline conditions, while altered levels of AtPQL1a severely reduced development of lateral roots in all conditions. This study provides the first step toward understanding the function of PQL proteins in plants and the role of NSCC in salinity tolerance.

The Use of High-Throughput Phenotyping for Assessment of Heat Stress-Induced Changes in Arabidopsis.

The worldwide rise in heatwave frequency poses a threat to plant survival and productivity. Determining the new marker phenotypes that show reproducible response to heat stress and contribute to heat stress tolerance is becoming a priority. In this study, we describe a protocol focusing on the daily changes in plant morphology and photosynthetic performance after exposure to heat stress using an automated noninvasive phenotyping system. Heat stress exposure resulted in an acute reduction of the quantum yield of photosystem II and increased leaf angle. In longer term, the exposure to heat also affected plant growth and morphology. By tracking the recovery period of the WT and mutants impaired in thermotolerance (hsp101), we observed that the difference in maximum quantum yield, quenching, rosette size, and morphology. By examining the correlation across the traits throughout time, we observed that early changes in photochemical quenching corresponded with the rosette size at later stages, which suggests the contribution of quenching to overall heat tolerance. We also determined that 6 h of heat stress provides the most informative insight in plant's responses to heat, as it shows a clear separation between treated and nontreated plants as well as the WT and hsp101. Our work streamlines future discoveries by providing an experimental protocol, data analysis pipeline, and new phenotypes that could be used as targets in thermotolerance screenings.

Diverse Traits Contribute to Salinity Tolerance of Wild Tomato Seedlings from the Galapagos Islands.

Traits of modern crops have been heavily selected in agriculture, leaving commercial lines often more susceptible to harsh conditions compared with their wild relatives. Understanding the mechanisms of stress tolerance in wild relatives can enhance crop performance under stress conditions such as high salinity. In this study, we investigated salinity tolerance of two species of wild tomato endemic to the Galapagos Islands, Solanum cheesmaniae and Solanum galapagense Since these tomatoes grow well despite being constantly splashed with seawater, they represent a valuable genetic resource for improving salinity tolerance in commercial tomatoes. To explore their potential, we recorded over 20 traits reflecting plant growth, physiology, and ion content in 67 accessions and two commercial tomato lines of Solanum lycopersicum. Salt treatments were applied for 10 d using supported hydroponics. The Galapagos tomatoes displayed greater tolerance to salt stress than the commercial lines and showed substantial natural variation in their responses. The accessions LA0317, LA1449, and LA1403 showed particularly high salinity tolerance based on growth under salinity stress. Therefore, Galapagos tomatoes should be further explored to identify the genes underlying their high tolerance and be used as a resource for increasing the salinity tolerance of commercial tomatoes. The generated data, along with useful analysis tools, have been packaged and made publicly available via an interactive online application (https://mmjulkowska.shinyapps.io/La_isla_de_tomato/) to facilitate trait selection and the use of Galapagos tomatoes for the development of salt-tolerant commercial tomatoes.