Plant cell surface pattern recognition receptors (PRRs) perceive non- or altered-self elicitors to induce immune responses. PRRs relay information across the plasma membrane and trigger downstream signalling via receptor-like cytoplasmic kinases such as BOTRYTIS-INDUCED KINASE 1 (BIK1). BIK1 associates with several PRRs and acts as a key executor of immune responses through the phosphorylation of substrate proteins. However, a comprehensive understanding of how BIK1 targets specific substrates and a full repertoire of these substrates are lacking. Here we defined the substrate specificity of BIK1 and used these data to predict candidate substrates in Arabidopsis. Using high-throughput biochemical and genetic screening of these candidates, we confirmed many as direct BIK1 substrates in vitro and novel regulators of plant immunity. Among the BIK1 substrates identified are MULTIPLE C2 DOMAIN AND TRANSMEMBRANE REGION PROTEIN 3, which we reveal regulates flagellin 22 (flg22)-induced plasmodesmata closure and immunity, and members of the largely uncharacterized CYCLIN-DEPENDENT KINASE-LIKE family, which we uncover as novel negative regulators of immunity. In parallel, we interrogated intracellular NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT (NLR) immune receptors for potential BIK1 phosphorylation motifs and identified multiple NLRs as direct BIK1 substrates. We reveal that BIK1 phosphorylation regulates NLR oligomerization, thus controlling a key activation step for these immune receptors. Together, our unbiased biochemical screens shed light on the central role of BIK1 as a key kinase shaping multiple layers of plant immune signalling.

Wheat is a major staple crop for over one-third of the world's population, crucial for global food security, economic stability and cultural traditions. Recently, single-cell and spatial omics approaches have transformed biological discovery, primarily in medical and animal sciences, and they are now beginning to be applied in plant research. Here we summarize the technical innovations and feasibility of spatial omics applications in wheat research, particularly for understanding developmental and environmental responses, thereby potentially enhancing wheat breeding. We highlight how these tools can reveal spatial and temporal patterns in gene expression, cellular heterogeneity and tissue organization in wheat. Furthermore, we propose developing a spatially resolved single-cell atlas of wheat across its life cycle to facilitate breakthroughs in basic research and potential applications in breeding. To achieve these goals, we advocate for a Wheat Spatial Omics Consortium to foster worldwide collaboration for overcoming barriers and developing sustainable and climate-resilient wheat.

Plant cells are connected to their neighbors via plasmodesmata facilitating the exchange of nutrients and signaling molecules. During immune responses, plasmodesmata close, but how this contributes towards a full immune response is unknown. To investigate this, we develop two transgenic lines which allow to induce plasmodesmal closure independently of immune elicitors, using the over-active CALLOSE SYNTHASE3 allele icals3m and the C-terminus of PDLP1 to drive callose deposition at plasmodesmata. Induction of plasmodesmal closure increases the expression of stress responsive genes, salicylic acid accumulation and resistance to Pseudomonas syringae DC3000. More homogeneous plasmodesmal closure using icals3m also leads to the accumulation of starch and sugars, decreases leaf growth, as well as hypersusceptibility to Botrytis cinerea. Based on the profile of responses, we conclude that plasmodesmal closure activates stress signaling, raising questions about the signals mediating this response and whether these responses occur in all circumstances when plasmodesmata close.

Chloroplasts act as environmental sensors, enabling rapid plant stress responses through operational retrograde signaling. While these signals operate on minutes-to-hours' time scales, their cumulative impact and function across a plant's lifecycle and in field conditions remains unknown. We investigated if retrograde signaling's transient changes to gene expression have cumulative effects by generating wheat mutants with primed SAL1-PAP retrograde pathway responsiveness. After confirming changes in photosynthesis and drought resilience under controlled conditions, we conducted 15 field trials across South-Eastern Australia, spanning diverse environments with varying temperatures, rainfall, and light conditions, analysing physiological responses, yield, biomass, and water productivity. We found gene locus-specific effects on biomass, yield, and water productivity. Multi-environment analysis showed the SAL-4A locus was more strongly associated with improved performance, with the converse for SAL-5D. Significantly, modulation of specific SAL subtypes enhanced photosynthetic efficiency and stress resilience while improving average yields by 4 and 8% respectively for 4A loci across 15 field sites, challenging the traditional yield-resilience trade-off paradigm. This study reveals retrograde signals operate in field environments, integrating environmental information across a plant's lifecycle to improve yield, water productivity and dynamic acclimation to diverse growing seasons, and highlight the importance of multi-environment field validation for crop modifications. ### Competing Interest Statement The authors have declared no competing interest.

Cellular responses to abiotic stress involve multiple signals such as reactive oxygen species (ROS), Ca2+, abscisic acid (ABA), and chloroplast-to-nucleus retrograde signals such as 3 '-phosphoadenosine 5 '-phosphate (PAP). The mechanism(s) by which these messengers intersect for cell regulation remain enigmatic, as do the roles of retrograde signals in specialized cells. Here we demonstrate a mechanistic link enabling ABA and PAP to coordinate chloroplast and plasma membrane ROS production. Contrary to its role in upregulating processes leading to quenching of ROS in foliar tissue, we show that in guard cells, PAP induces chloroplast ROS accumulation via photosynthetic electron transport and apoplast ROS via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) proteins. Both subcellular ROS sources are necessary for stress hormone ABA-mediated stomatal closure, as well as PAP-mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD instead of RBOHF. Three calcium-dependent protein kinases (CPKs) transcriptionally induced by PAP, namely CPK13, CPK32, and CPK34, concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two SLAC1 serine (S) residues, including S120, which is also targeted by the ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpression of the PAP-induced CPKs rescues stomatal closure in ost1. Our data identify chloroplast retrograde signals as critical nodes in cellular stress response networks of guard cells.

Chloroplast-to-nucleus retrograde signalling enables rapid stress responses in plants, but whether these signals accumulate to affect crop performance across entire growing seasons under field conditions remains unknown. We generated wheat mutants with targeted deletions in specific SAL gene copies from two distinct homeologous groups (TaSAL1 and TaSAL2), creating lines with enhanced stress signal responsiveness. We tested these lines across 15 field trials spanning diverse Australian environments with varying temperatures, rainfall, and irrigation regimes, measuring physiological responses, yield, biomass, and water productivity. Lines with TaSAL2 gene deletions showed 4-8% yield improvements with enhanced water productivity, while TaSAL1 deletions reduced yields. The TaSAL2 mutants maintained superior photosynthetic function under drought stress, showed improved relative water content, and demonstrated enhanced yield stability across environments. Canopy temperature measurements revealed dynamic stomatal regulation, with increased closure during midday stress periods but normal aperture under benign conditions. Significantly, specific SAL modifications enhanced photosynthetic efficiency and stress resilience without traditional yield penalties. Targeted modification of specific SAL homeologous groups can simultaneously improve both yield and stress tolerance in wheat. This demonstrates that retrograde signalling integrates environmental information across the plant lifecycle and highlights the importance of locus-specific targeting and multienvironment field validation for crop modifications.

Plasmodesmata are plasma membrane-lined connections that join plant cells to their neighbours, establishing an intercellular cytoplasmic continuum through which molecules can travel between cells, tissues, and organs. As plasmodesmata connect almost all cells in plants, their molecular traffic carries information and resources across a range of scales, but dynamic control of plasmodesmal aperture can change the possible domains of molecular exchange under different conditions. Plasmodesmal aperture is controlled by specialised signalling cascades accommodated in spatially discrete membrane and cell wall domains. Thus, the composition of plasmodesmata defines their capacity for molecular trafficking. Further, their shape and density can likewise define trafficking capacity, with the cell walls between different cell types hosting different numbers and forms of plasmodesmata to drive molecular flux in physiologically important directions. The molecular traffic that travels through plasmodesmata ranges from small metabolites through to proteins, and possibly even larger mRNAs. Smaller molecules are transmitted between cells via passive mechanisms but how larger molecules are efficiently trafficked through plasmodesmata remains a key question in plasmodesmal biology. How plasmodesmata are formed, the shape they take, what they are made of, and what passes through them regulate molecular traffic through plants, underpinning a wide range of plant physiology.

Cellular responses to abiotic stress involve multiple signals including secondary messengers such as reactive oxygen species (ROS) and Ca2+, phytohormones such as abscisic acid (ABA) and chloroplast-to-nucleus retrograde signals such as 3’-phosphoadenosine 5’-phosphate (PAP). Mechanism(s) by which these messengers, produced in different subcellular compartments, intersect for cell regulation remain enigmatic. Previously we showed that the chloroplast retrograde signal PAP, similar to ABA, induces an increase in ROS levels in guard cells ([Pornsiriwong et al , 2017][1]). Here we demonstrate a mechanistic link enabling ABA and PAP to coordinate both chloroplast and plasma membrane ROS production. In whole leaves, PAP alters various ROS-related processes including plasmodesmal permeability as well as responses to ozone and the bacterial elicitor flg22, but mainly initiates processes that quench ROS during oxidative stress. Conversely, we show in guard cells, both PAP and ABA induce an increase in ROS levels in both chloroplasts via photosynthetic electron transport, and the apoplast via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). Both subcellular ROS sources were necessary for ABA- and PAP-mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD, instead of RBOHF, for apoplastic ROS mediated stomatal closure. We identified three calcium-dependent protein kinases (CPKs) as the post-translational activators of RBOHD-mediated ROS production. CPK13, CPK32, and CPK34 were transcriptionally induced by PAP and concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two Serine (S) residues, including S120 which is also targeted by the core ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpression of the PAP-induced CPKs rescues stomatal closure in ost1. Our data identify stomatal chloroplasts, to be nodes in the multifaceted cellular stress response networks as they are both sources and mediators of ROS and retrograde signals such PAP. Thus, chloroplasts are not just mediators of photosynthesis in response to, for example, excess light, but can serve as critical nodes in the multifaceted cellular stress response networks in specialized cells via retrograde signals, providing support to the concept of sensory plastids.Significance Statement The chloroplast is an environmental sensor for stresses such as excess light and drought via the activation of photosynthetic-mediated retrograde signals. However, how does it function in specialized cells for which carbon fixation is secondary? Here we show the chloroplast is an important node to coordinate multiple plant signaling pathways in response to stresses such as drought. The chloroplast retrograde signal 3’-phosphoadenosine 5’-phosphate (PAP) plays multiple roles in reactive oxygen species (ROS) signaling and homeostasis. While PAP suppresses ROS in photosynthetic tissue, PAP instead induces guard cell ROS in chloroplasts and extracellular space to induce stomatal closure. We decipher how PAP-induced proteins activate both extracellular ROS production and anion channels for stomatal closure, thus providing a mechanism by which chloroplasts provide a strategic complement to canonical hormonal pathways in regulating plant physiological responses in specialized cells.### Competing Interest StatementThe authors have declared no competing interest. [1]: #ref-58

The plant immune system relies on the perception of molecules that signal the presence of a microbe threat. This triggers signal transduction that mediates a range of cellular responses via a collection of molecular machinery including receptors, small molecules, and enzymes. One response to pathogen perception is the restriction of cell-to-cell communication by plasmodesmal closure. We previously found that while chitin and flg22 trigger specialized immune signaling cascades in the plasmodesmal plasma membrane, both execute plasmodesmal closure via callose synthesis at the plasmodesmata. Therefore, the signaling pathways ultimately converge at or upstream of callose synthesis. To establish the hierarchy of signaling at plasmodesmata and characterize points of convergence in microbe elicitor-triggered signaling, we profiled the dependence of plasmodesmal responses triggered by different elicitors on a range of plasmodesmal signaling machinery. We identified that, like chitin, flg22 signals via RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) to induce plasmodesmal closure. Further, we found that PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1), PDLP5, and CALLOSE SYNTHASE 1 (CALS1) are common to microbe-and salicylic acid (SA)-triggered responses, identifying PDLPs as a candi-date signaling nexus. To understand how PDLPs relay a signal to CALS1, we screened for PDLP5 interactors and found NON-RACE SPECIFIC DISEASE RESISTANCE/ HIN1 HAIRPIN-INDUCED-LIKE protein 3 (NHL3), which is also required for chitin-, flg22-and SA-triggered plasmodesmal responses and PDLP-mediated acti-vation of callose synthesis. We conclude that a PDLP-NHL3 complex acts as an integrating node of plasmodesmal signaling cascades, transmitting multiple immune signals to activate CALS1 and plasmodesmata closure.

Plant cells are connected by cytoplasmic bridges called plasmodesmata. Plasmodesmata are lined by the plasma membrane, essentially forming tunnels that directly connect the cytoplasm of adjacent cells through which soluble molecules can move from cell to cell. This cell-to-cell mobility is underpinned by cytoplasmic advection and diffusion in a manner dependent on molecular size. This movement of molecules is regulated by the aperture of plasmodesmata. GREEN FLUORESCENT PROTEIN (GFP) is a 27 kDa soluble protein that can move passively between cells via plasmodesmata. Thus, it serves as an ideal probe to assess plasmodesmal aperture. GFP can be transgenically produced in single cells by microprojectile bombardment-mediated transformation, and its cell-to-cell mobility can be measured by live-cell imaging and counting the number of cells (or cell layers) to which it has moved. Thus, the number of cells in which GFP is visible serves as a measure of plasmodesmal aperture and functional cell-to-cell connectivity. Here we present methods for microprojectile bombardment of GFP into leaf epidermal cells and statistical analysis of resulting data.

Photosystem biogenesis in the chloroplast requires a concerted effort between synthesis and assembly of components including protein subunits, pigments, and other cofactors that varies both temporally and spatially. [ Sun et al. (2019) ][1] investigate the translation zone (T-zone) of unicellular

Protocol for recombinant protein expression in E. coli for protein purification and subsequent enzyme assays, protein crystallography etc.

Chloroplast retrograde signaling networks are vital for chloroplast biogenesis, operation, and signaling, including excess light and drought stress signaling. To date, retrograde signaling has been considered in the context of land plant adaptation, but not regarding the origin and evolution of signaling cascades linking chloroplast function to stomatal regulation. We show that key elements of the chloroplast retrograde signaling process, the nucleotide phosphatase (SAL1) and 3'-phosphoadenosine-5'-phosphate (PAP) metabolism, evolved in streptophyte algae-the algal ancestors of land plants. We discover an early evolution of SAL1-PAP chloroplast retrograde signaling in stomatal regulation based on conserved gene and protein structure, function, and enzyme activity and transit peptides of SAL1s in species including flowering plants, the fern Ceratopteris richardii, and the moss Physcomitrella patens. Moreover, we demonstrate that PAP regulates stomatal closure via secondary messengers and ion transport in guard cells of these diverse lineages. The origin of stomata facilitated gas exchange in the earliest land plants. Our findings suggest that the conquest of land by plants was enabled by rapid response to drought stress through the deployment of an ancestral SAL1-PAP signaling pathway, intersecting with the core abscisic acid signaling in stomatal guard cells.

Chloroplast development in germinating seedlings initiates upon illumination. Whereas chloroplasts in true leaves develop directly from proplastids, chloroplasts in cotyledons of dark-grown seedlings develop from an intermediate type of plastid called an etioplast. During development, etioplasts

Photosystem biogenesis in the chloroplast requires a concerted effort between synthesis and assembly of components including protein subunits, pigments, and other cofactors that varies both temporally and spatially. [ Sun et al. (2019) ][1] investigate the translation zone (T-zone) of unicellular

Identifying natural genetic variation, understanding how it influences traits, and utilizing it for crop improvement is a major objective in plant science. [ Miller et al. (2019) ][1] have identified genetic variation in the promoter region of BnaUPL3.C03 from a panel of Brassica napus accessions

Numerous proteins coordinate the synthesis, delivery, and assembly of cell wall components during cell wall biosynthesis ([McFarlane et al., 2014][1]). KORRIGAN1 (KOR1) is a membrane-anchored endo-β1,4-glucanase glycoprotein involved in the cellulose biosynthesis of primary and secondary cell walls

Protocol for generating chemically compotent (ie. for heat shock transformation) E. coli cells for transformation (e.g. heterologous protein expression, cloning).

Most proteins within a cell are encoded in the nucleus and then translated in the cytosol, but how do they end up where they need to be? With the exception of the few proteins expressed within the chloroplast, the process of shipping nucleus-encoded proteins into the chloroplast is dependent on N-