Fluorescent biosensors are powerful tools for tracking analytes or cellular processes in live organisms and allowing visualization of the spatial and temporal dynamics of cellular regulators. Fluorescent protein (FP)based biosensors are extensively employed due to their high selectivity and low invasiveness. A variety of FP-based biosensors have been engineered and applied in plant research to visualize dynamic changes in pH, redox state, concentration of molecules (ions, sugars, peptides, ATP, reactive oxygen species, and phytohormones), and activity of transporters. In this chapter, we briefly summarize reported uses of FP-based biosensors in planta and show simple methods to monitor the dynamics of intracellular Ca2+ in Arabidopsis thaliana using a ratiometric genetically encoded Ca2+ indicator, MatryoshCaMP6s.

The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a significant role in plastid-to-nucleus retrograde signaling in plants to ensure that nuclear gene expression is attuned to the needs of the chloroplast. Well-known sites of synthesis of chlorophyll for photosynthesis, plant chloroplasts also export heme and heme-derived linear tetrapyrroles (bilins), two critical metabolites respectively required for essential cellular activities and for light sensing by phytochromes. Here we establish that Chlamydomonas reinhardtii, one of many chloro-phyte species that lack phytochromes, can synthesize bilins in both plastid and cytosol compartments. Genetic analyses show that both pathways contribute to iron acquisition from extracellular heme, whereas the plastid-localized pathway is essential for light-dependent greening and phototrophic growth. Our discovery of a bilin-dependent nuclear gene network implicates a widespread use of bilins as retrograde signals in oxygenic photosynthetic species. Our studies also suggest that bilins trigger critical metabolic pathways to detoxify molecular oxygen produced by photosynthesis, thereby permitting survival and phototrophic growth during the light period.

CRISPR-Cas genetic engineering of plants holds tremendous potential for providing food security, battling biotic and abiotic crop stresses caused by climate change, and for environmental remediation and sustainability. Since the discovery of CRISPR-Cas technology, its usefulness has been demonstrated widely, including for genome editing in plants. Despite the revolutionary nature of genome-editing tools and the notable progress that these tools have enabled in plant genetic engineering, there remain many challenges for CRISPR applications in plant biotechnology. Nanomaterials could address some of the most critical challenges of CRISPR genome editing in plants through improvements in cargo delivery, species independence, germline transformation and gene editing efficiency. This Perspective identifies major barriers preventing CRISPR-mediated plant genetic engineering from reaching its full potential, and discusses ways that nanoparticle technologies can lower or eliminate these barriers. We also describe advances that are needed in nanotechnology to facilitate and accelerate plant genome editing. Timely advancement of the application of CRISPR technologies in plant engineering is crucial for our ability to feed and sustain the growing human population under a changing global climate.

The most recent GreenCut (GreenCut2) represents a collection of 597 proteins in Chlamydomonas and other green lineage photosynthetic organisms, but not in non -photosynthetic (heterotrophic) organisms. GreenCut2 proteins have diverse functions, with many involved in chloroplast processes including photosynthesis and various biosynthetic pathways. GreenCut2 proteins also have roles in assembly and maintenance of chloroplasts, as well as in plant and algal regulatory processes. This chapter focuses on how GreenCut2 bioinformatic analyses were performed, discusses new insights into GreenCut2 proteins that have recently been characterized, suggests new ways to group these proteins based on their known or inferred biological functions, and re-examines potential functions of some GreenCut2 'unknowns' for which there is now experimental information.

To investigate the dynamics of photosynthetic pigment-protein complexes in vascular plants at high resolution in an aqueous environment, membrane-protruding oxygen-evolving complexes (OECs) associated with photosystem II (PSII) on spinach (Spinacia oleracea) grana membranes were examined using contact mode atomic force microscopy. This study represents, to our knowledge, the first use of atomic force microscopy to distinguish the putative large extrinsic loop of Photosystem II CP47 reaction center protein (CP47) from the putative oxygen-evolving enhancer proteins 1, 2, and 3 (PsbO, PsbP, and PsbQ) and large extrinsic loop of Photosystem II CP43 reaction center protein (CP43) in the PSII-OEC extrinsic domains of grana membranes under conditions resulting in the disordered arrangement of PSII-OEC particles. Moreover, we observed uncharacterized membrane particles that, based on their physical characteristics and electrophoretic analysis of the polypeptides associated with the grana samples, are hypothesized to be a domain of photosystem I that protrudes from the stromal face of single thylakoid bilayers. Our results are interpreted in the context of the results of others that were obtained using cryo-electron microscopy (and single particle analysis), negative staining and freeze-fracture electron microscopy, as well as previous atomic force microscopy studies.

Glutamate has dual roles in metabolism and signaling; thus, signaling functions must be isolatable and distinct from metabolic fluctuations, as seen in low-glutamate domains at synapses. In plants, wounding triggers electrical and calcium (Ca2+) signaling, which involve homologs of mammalian glutamate receptors. The hydraulic dispersal and squeeze-cell hypotheses implicate pressure as a key component of systemic signaling. Here, we identify the stretch-activated anion channel MSL10 as necessary for proper wound-induced electrical and Ca2+ signaling. Wound gene induction, genetics, and Ca2+ imaging indicate that MSL10 acts in the same pathway as the glutamate receptor-like proteins (GLRs). Analogous to mammalian NMDA glutamate receptors, GLRs may serve as coincidence detectors gated by the combined requirement for ligand binding and membrane depolarization, here mediated by stretch activation of MSL10. This study provides a molecular genetic basis for a role of mechanical signal perception and the transmission of long-distance electrical and Ca2+ signals in plants.

Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases Cr Delta 4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.

Mechanical stress influences cell-and tissue-scale processes across all kingdoms. It remains challenging to delineate how mechanical stress, originating at these different length scales, impacts cell and tissue form. We combine growth tracking of cells, quantitative image analysis, as well as molecular and mechanical perturbations to address this problem in pavement cells of Arabidopsis thaliana cotyledon tissue. We show that microtubule organization based on chemical signals and cell-shape-derived mechanical stress varies during early stages of pavement cell development and is mediated by the evolutionary conserved proteins, KATANIN and CLASP. However, we find that these proteins regulate microtubule organization in response to tissue-scale mechanical stress to different extents in the cotyledon epidermis. Our results further demonstrate that regulation of cotyledon form is uncoupled from the mechanical-stress-dependent control of pavement cell shape that relies on microtubule organization governed by subcellular mechanical stress.

As scientists, we are at least as excited about the open questions-the things we do not know-as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such "rules" conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring.