Proteome profiling of vascular sap regarding Eucalyptus grandis, Eucalyptus urophylla, and Eucalyptus camaldulensis.

The plant vascular system is a key element for long-distance communication. Understanding its composition may provide valuable information on how plants grow and develop themselves. In this study, a quantitative proteome dataset of the vascular sap proteome of three commercially important Eucalyptus species was shown. Protein extraction was carried out using a pressure bomb, whereas only in silico predicted extracellular proteins were considered as part of the sap proteome. A total of 132 different proteins were identified in all three Eucalyptus species and the most abundant proteome subset within all three species was comprised of proteins involved in the carbohydrate metabolic process, proteolysis, components of membrane, and defense response. The sap proteome of the species E. grandis and E. urophylla revealed the highest similarities. Functional classification indicated that the sap proteome of E. grandis and E. urophylla are mostly comprised of proteins involved in defense response and proteolysis; whereas no prominent functional class was observed for the E. camaldulensis species. Quantitative comparison highlighted characteristic sap proteins in each of the Eucalyptus species. The results that could be found in this study can be used as a reference for the proteome sap analysis of Eucalyptus plants grown under different conditions.

The role of long-distance mobile metabolites in the plant stress response and signaling.

Plants developed sophisticated mechanisms to perceive environmental stimuli and generate appropriate signals to maintain optimal growth and stress responses. A fascinating strategy employed by plants is the use of long-distance mobile signals which can trigger local and distant responses across the entire plant. Some metabolites play a central role as long-distance mobile signals allowing plants to communicate across tissues and mount robust stress responses. In this review, we summarize the current knowledge regarding the various long-distance mobile metabolites and their functions in stress response and signaling pathways. We also raise questions with respect to how we can identify new mobile metabolites and engineer them to improve plant health and resilience.

Rapid Propagation of Ca2+ Waves and Electrical Signals in the Liverwort Marchantia polymorpha.

In response to both biotic and abiotic stresses, vascular plants transmit long-distance Ca2+ and electrical signals from localized stress sites to distant tissues through their vasculature. Various models have been proposed for the mechanisms underlying the long-distance signaling, primarily centered around the presence of vascular bundles. We here demonstrate that the non-vascular liverwort Marchantia polymorpha possesses a mechanism for propagating Ca2+ waves and electrical signals in response to wounding. The propagation velocity of these signals was approximately 1-2 mm s-1, equivalent to that observed in vascular plants. Both Ca2+ waves and electrical signals were inhibited by La3+ as well as tetraethylammonium chloride, suggesting the crucial importance of both Ca2+ channel(s) and K+ channel(s) in wound-induced membrane depolarization as well as the subsequent long-distance signal propagation. Simultaneous recordings of Ca2+ and electrical signals indicated a tight coupling between the dynamics of these two signaling modalities. Furthermore, molecular genetic studies revealed that a GLUTAMATE RECEPTOR-LIKE (GLR) channel plays a central role in the propagation of both Ca2+ waves and electrical signals. Conversely, none of the three two-pore channels were implicated in either signal propagation. These findings shed light on the evolutionary conservation of rapid long-distance Ca2+ wave and electrical signal propagation involving GLRs in land plants, even in the absence of vascular tissue.

A cucumber protein, Phloem Phosphate Stress-Repressed 1, rapidly degrades in response to a phosphate stress condition.

Under depleted external phosphate (Pi), many plant species adapt to this stress by initiating downstream signaling cascades. In plants, the vascular system delivers nutrients and signaling agents to control physiological and developmental processes. Currently, limited information is available regarding the direct role of phloem-borne long-distance signals in plant growth and development under Pi stress conditions. Here, we report on the identification and characterization of a cucumber protein, Cucumis sativus Phloem Phosphate Stress-Repressed 1 (CsPPSR1), whose level in the phloem translocation stream rapidly responds to imposed Pi-limiting conditions. CsPPSR1 degradation is mediated by the 26S proteasome; under Pi-sufficient conditions, CsPPSR1 is stabilized by its phosphorylation within the sieve tube system through the action of CsPPSR1 kinase. Further, we discovered that CsPPSR1 kinase was susceptible to Pi starvation-induced degradation in the sieve tube system. Our findings offer insight into a molecular mechanism underlying the response of phloem-borne proteins to Pi-limited stress conditions.

NRAMP6 and NRAMP1 cooperatively regulate root growth and manganese translocation under manganese deficiency in Arabidopsis.

The essential micronutrient manganese (Mn) in plants regulates multiple biological processes including photosynthesis and oxidative stress. Some Natural Resistance-Associated Macrophage Proteins (NRAMPs) have been reported to play critical roles in Mn uptake and reutilization in low Mn conditions. NRAMP6 was demonstrated to regulate cadmium tolerance and iron utilization in Arabidopsis. Nevertheless, it is unclear whether NRAMP6 plays a role in Mn nutrition. Here, we report that NRAMP6 cooperates with NRAMP1 in Mn utilization. Mutation of NRAMP6 in nramp1 but not in a wild-type background reduces root growth and Mn translocation from the roots to shoots under Mn deficient conditions. Grafting experiments revealed that NRAMP6 expression in both the roots and shoots is required for root growth and Mn translocation under Mn deficiency. We also showed that NRAMP1 could replace NRAMP6 to sustain root growth under Mn deficiency, but not vice versa. Mn deficiency does not affect the transcript level of NRAMP6, but is able to increase and decrease the protein accumulation of NRAMP6 in roots and shoots, respectively. Furthermore, NRAMP6 can be localized to both the plasma membrane and endomembranes including the endoplasmic reticulum, and Mn deficiency enhances the localization of NRAMP6 to the plasma membrane in Arabidopsis plants. NRAMP6 could rescue the defective growth of the yeast mutant Δsmf2, which is deficient in endomembrane Mn transport. Our results reveal the important role of NRAMP6 in Mn nutrition and in the long-distance signaling between the roots and shoots under Mn deficient conditions.

Rhizosphere microbiome mediates systemic root metabolite exudation by root-to-root signaling.

Microbial communities associated with roots confer specific functions to their hosts, thereby modulating plant growth, health, and productivity. Yet, seminal questions remain largely unaddressed including whether and how the rhizosphere microbiome modulates root metabolism and exudation and, consequently, how plants fine tune this complex belowground web of interactions. Here we show that, through a process termed systemically induced root exudation of metabolites (SIREM), different microbial communities induce specific systemic changes in tomato root exudation. For instance, systemic exudation of acylsugars secondary metabolites is triggered by local colonization of bacteria affiliated with the genus Bacillus Moreover, both leaf and systemic root metabolomes and transcriptomes change according to the rhizosphere microbial community structure. Analysis of the systemic root metabolome points to glycosylated azelaic acid as a potential microbiome-induced signaling molecule that is subsequently exuded as free azelaic acid. Our results demonstrate that rhizosphere microbiome assembly drives the SIREM process at the molecular and chemical levels. It highlights a thus-far unexplored long-distance signaling phenomenon that may regulate soil conditioning.

Integration of Electrical Signals and Phytohormones in the Control of Systemic Response.

Plants are constantly exposed to environmental stresses. Local stimuli sensed by one part of a plant are translated into long-distance signals that can influence the activities in distant tissues. Changes in levels of phytohormones in distant parts of the plant occur in response to various local stimuli. The regulation of hormone levels can be mediated by long-distance electrical signals, which are also induced by local stimulation. We consider the crosstalk between electrical signals and phytohormones and identify interaction points, as well as provide insights into the integration nodes that involve changes in pH, Ca2+ and ROS levels. This review also provides an overview of our current knowledge of how electrical signals and hormones work together to induce a systemic response.

Tandem Expression of a Mobile RNA and Its RNA-Binding Protein(s) Enhances Tuber Productivity in Potato.

A significant number of discoveries in past two decades have established the importance of long-distance signaling in controlling plant growth, development, and biotic and abiotic stress responses. Numerous mobile signals, such as mRNAs, proteins, including RNA-binding proteins, small RNAs, sugars, and phytohormones, are shown to regulate various agronomic traits such as flowering, fruit, seed development, and tuberization. Potato is a classic model tuber crop, and several mobile signals are known to govern tuber development. However, it is unknown if these mobile signals have any synergistic effects on potato crop improvement. Here, we employed a simple innovative strategy to test the cumulative effects of a key mobile RNA, StBEL5, and its RNA-binding proteins, StPTB1, and -6 on tuber productivity of two potato cultivars, Solanum tuberosum cv. Désirée and subspecies andigena, using a multi-gene stacking approach. In this approach, the coding sequences of StBEL5 and StPTB1/6 are driven by their respective native promoters to efficiently achieve targeted expression in phloem for monitoring tuber productivity. We demonstrate that this strategy resulted in earliness for tuberization and enhanced tuber productivity by 2-4 folds under growth chamber, greenhouse, and field conditions. This multi-gene stacking approach could be adopted to other crops, whose agronomic traits are governed by mobile macromolecules, expanding the possibilities to develop crops with improved traits and enhanced yields.

Cytokinin biosynthesis and transport for systemic nitrogen signaling.

The plasticity of growth and development in response to environmental changes is one of the essential aspects of plant behavior. Cytokinins play an important role as signaling molecules in the long-distance communication between organs in systemic growth regulation in response to nitrogen. The spatial distribution of the expression sites of cytokinin biosynthesis genes leads to structural differences in the molecular species transported through the xylem and phloem, giving root-borne trans-hydroxylated cytokinins, namely trans-zeatin (tZ) type, a specialized efficacy in regulating shoot growth. Furthermore, root-to-shoot translocation via the xylem, tZ, and its precursor, the tZ riboside, controls different sets of shoot growth traits to fine-tune shoot growth in response to nitrogen availability. In addition to nitrogen, photosynthetically generated sugars positively regulate de novo cytokinin biosynthesis in the roots, and contribute to plant growth under elevated CO2 conditions. In shoot-to-root signaling, cytokinins also play a role in the regulation of nutrient acquisition and root system growth in cooperation with other types of signaling molecules, such as C-TERMINALLY ENCODED PEPTIDE DOWNSTREAMs. As cytokinin is a key regulator for the maintenance of shoot apical meristem, deepening our understanding of the regulatory mechanisms of cytokinin biosynthesis and transport in response to nitrogen is important not only for basic comprehension of plant growth, but also to ensure the stability of agricultural production.

Systemic Regulation of Iron Acquisition by Arabidopsis in Environments with Heterogeneous Iron Distributions.

Nutrient distribution within the soil is generally heterogeneous. Plants, therefore, have evolved sophisticated systemic processes enabling them to optimize their nutrient acquisition efficiency. By organ-to-organ communication in Arabidopsis thaliana, for instance, iron (Fe) starvation in one part of a root drives the upregulation of a high-affinity Fe-uptake system in other root regions surrounded by sufficient levels of Fe. This compensatory response through Fe-starvation-triggered organ-to-organ communication includes the upregulation of Iron-regulated transporter 1 (IRT1) gene expression on the Fe-sufficient side of the root; however, the molecular basis underlying this long-distance signaling remains unclear. Here, we analyzed gene expression by RNA-seq analysis of Fe-starved split-root cultures. Genome-wide expression analysis showed that localized Fe depletion in roots upregulated several genes involved in Fe uptake and signaling, such as IRT1, in a distant part of the root exposed to Fe-sufficient conditions. This result indicates that long-distance signaling for Fe demand alters the expression of a subset of genes responsible for Fe uptake and coumarin biosynthesis to maintain a level of Fe acquisition sufficient for the entire plant. Loss of IRON MAN/FE-UPTAKE-INDUCING PEPTIDE (IMA/FEP) leads to the disruption of compensatory upregulation of IRT1 in the root surrounded by sufficient Fe. In addition, our split-root culture-based analysis provides evidence that the IMA3/FEP1-MYB10/72 pathway mediates long-distance signaling in Fe homeostasis through the regulation of coumarin biosynthesis. These data suggest that the signaling of IMA/FEP, a ubiquitous family of metal-binding peptides, is critical for organ-to-organ communication in response to Fe starvation under heterogeneous Fe conditions in the surrounding environment.

Plant gene silencing signals move from the phloem to influence gene expression in shoot apical meristems.

BACKGROUND: Small RNAs (sRNA) are potent regulators of gene expression that can diffuse short distances between cells and move long distances through plant vasculature. However, the degree to which sRNA silencing signals can move from the phloem to the shoot apical meristem (SAM) remains unclear. RESULTS: Two independent transgenic approaches were used to examine whether phloem sRNA silencing can reach different domains of the SAM and silence SAM-expressed genes. First, the phloem companion-cell specific SUCROSE-PROTON SYMPORTER2 (SUC2) promoter was used to drive expression of an inverted repeat to target the FD gene, an exclusively SAM-localized floral regulator. Second, the SUC2 promoter was used to express an artificial microRNA (aMiR) designed to target a synthetic CLAVATA3 (CLV3) transgene in SAM stem cells. Both phloem silencing signals phenocopied the loss of function of their targets and altered target gene expression suggesting that a phloem-to-SAM silencing communication axis exists, connecting distal regions of the plant to SAM stem cells. CONCLUSIONS: Demonstration of phloem-to-SAM silencing reveals a regulatory link between somatic sRNA expressed in distal regions of the plant and the growing shoot. Since the SAM stem cells ultimately produce the gametes, we discuss the intriguing possibility that phloem-to-SAM sRNA trafficking could allow transient somatic sRNA expression to manifest stable, transgenerational epigenetic changes.

Lotus japonicus HAR1 regulates root morphology locally and systemically under a moderate nitrate condition in the absence of rhizobia.

MAIN CONCLUSION: The local and long-distance signaling pathways mediated by the leucine-rich repeat receptor kinase HAR1 suppress root branching and promote primary root length in response to nitrate supply. The root morphology of higher plants changes plastically to effectively absorb nutrients and water from the soil. In particular, legumes develop root organ nodules, in which symbiotic rhizobia fix atmospheric nitrogen in nitrogen-poor environments. The number of nodules formed in roots is negatively regulated by a long-distance signaling pathway that travels through shoots called autoregulation of nodulation (AON). In the model plant Lotus japonicus, defects in AON genes, such as a leucine-rich repeat receptor kinase HYPERNODULATION ABERRANT ROOT FORMATION 1 (HAR1), an orthologue of CLAVATA1, and the F-box protein TOO MUCH LOVE (TML), induce the formation of an excess number of nodules. The loss-of-function mutant of HAR1 exhibits a short and bushy root phenotype in the absence of rhizobia. We show that the har1 mutant exhibits high nitrate sensitivity during root development. The uninfected har1 mutant significantly increased lateral root number and reduced primary root length in the presence of 3 mM nitrate, compared with the wild-type and tml mutant. Grafting experiments indicated that local and long-distance signaling pathways via root- and shoot-acting HAR1 additively regulated root morphology under the moderate nitrate concentrations. These findings allow us to propose that HAR1-mediated signaling pathways control the root system architecture by suppressing lateral root branching and promoting primary root elongation in response to nitrate availability.

Cadmium interference with iron sensing reveals transcriptional programs sensitive and insensitive to reactive oxygen species.

Iron (Fe) is an essential micronutrient whose uptake is tightly regulated to prevent either deficiency or toxicity. Cadmium (Cd) is a non-essential element that induces both Fe deficiency and toxicity; however, the mechanisms behind these Fe/Cd-induced responses are still elusive. Here we explored Cd- and Fe-associated responses in wild-type Arabidopsis and in a mutant that overaccumulates Fe (opt3-2). Gene expression profiling revealed a large overlap between transcripts induced by Fe deficiency and Cd exposure. Interestingly, the use of opt3-2 allowed us to identify additional gene clusters originally induced by Cd in the wild type but repressed in the opt3-2 background. Based on the high levels of H2O2 found in opt3-2, we propose a model where reactive oxygen species prevent the induction of genes that are induced in the wild type by either Fe deficiency or Cd. Interestingly, a defined cluster of Fe-responsive genes was found to be insensitive to this negative feedback, suggesting that their induction by Cd is more likely to be the result of an impaired Fe sensing. Overall, our data suggest that Fe deficiency responses are governed by multiple inputs and that a hierarchical regulation of Fe homeostasis prevents the induction of specific networks when Fe and H2O2 levels are elevated.

Encoding, transmission, decoding, and specificity of calcium signals in plants.

Calcium acts as a signal and transmits information in all eukaryotes. Encoding machinery consisting of calcium channels, stores, buffers, and pumps can generate a variety of calcium transients in response to external stimuli, thus shaping the calcium signature. Mechanisms for the transmission of calcium signals have been described, and a large repertoire of calcium binding proteins exist that can decode calcium signatures into specific responses. Whilst straightforward as a concept, mysteries remain as to exactly how such information processing is biochemically implemented. Novel developments in imaging technology and genetically encoded sensors (such as calcium indicators), in particular for multi-signal detection, are delivering exciting new insights into intra- and intercellular calcium signaling. Here, we review recent advances in characterizing the encoding, transmission, and decoding mechanisms, with a focus on long-distance calcium signaling. We present technological advances and computational frameworks for studying the specificity of calcium signaling, highlight current gaps in our understanding and propose techniques and approaches for unravelling the underlying mechanisms.

Micrografting device for testing systemic signaling in Arabidopsis.

Grafting techniques have been applied in studies of systemic, long-distance signaling in several model plants. Seedling grafting in Arabidopsis, known as micrografting, enables investigation of the molecular mechanisms of systemic signaling between shoots and roots. However, conventional micrografting requires a high level of skill, limiting its use. Thus, an easier user-friendly method is needed. Here, we developed a silicone microscaled device, the micrografting chip, to obviate the need for training and to generate less stressed and more uniformly grafted seedlings. The chip has tandemly arrayed units, each of which consists of a seed pocket for seed germination and a micro-path with pairs of pillars for hypocotyl holding. Grafting, including seed germination, micrografting manipulation and establishment of tissue reunion, is performed on the chip. Using the micrografting chip, we evaluated the effect of temperature and the carbon source on grafting, and showed that a temperature of 27°C and a sucrose concentration of 0.5% were optimal. We also used the chip to investigate the mechanism of systemic signaling of iron status using a quadruple nicotianamine synthase (nas) mutant. The constitutive iron-deficiency response in the nas mutant because of iron accumulation in shoots was significantly rescued by grafting of wild-type shoots or roots, suggesting that shoot- and root-ward translocation of nicotianamine-iron complexes and/or nicotianamine is essential for iron mobilization. Thus, our micrografting chip will promote studies of long-distance signaling in plants.

Glutamate-Induced Electrical and Calcium Signals in the Moss Physcomitrella patens.

The mode of transmission of signals between plant cells is an important aspect of plant physiology. The main role in the generation of long-distance signals is played by changes in the membrane potential and cytoplasm calcium concentration, but the relationship between these responses evoked by the same stimuli in the same plant remains unknown. As one of the first plants that colonized land, the moss Physcomitrella patens is a suitable model organism for studying the evolution of signaling pathways in plants. Here, by the application of glutamate as a stimulus, we demonstrated that electrical but not calcium signals can be true carriers of information in long-distance signaling in Physcomitrella. The generation of electrical signals in a form of propagating transient depolarization seems to be dependent on the opening of calcium channels since the responses were reduced or totally blocked by calcium channel inhibitors. While the microelectrode measurements demonstrated the transmission of electric signals between leaf cells and juvenile cells (protonema), the fluorescence imaging of cytoplasmic calcium changes indicated that calcium response occurs only locally-at the site of glutamate application, and only in protonema cells. This study indicates different involvement of glutamate-induced electrical and calcium signals in cell-to-cell communication in these evolutionarily old terrestrial plants.

Universal poroelastic mechanism for hydraulic signals in biomimetic and natural branches.

Plants constantly undergo external mechanical loads such as wind or touch and respond to these stimuli by acclimating their growth processes. A fascinating feature of this mechanical-induced growth response is that it can occur rapidly and at long distance from the initial site of stimulation, suggesting the existence of a fast signal that propagates across the whole plant. The nature and origin of the signal is still not understood, but it has been recently suggested that it could be purely mechanical and originate from the coupling between the local deformation of the tissues (bending) and the water pressure in the plant vascular system. Here, we address the physical origin of this hydromechanical coupling using a biomimetic strategy. We designed soft artificial branches perforated with longitudinal liquid-filled channels that mimic the basic features of natural stems and branches. In response to bending, a strong overpressure is generated in the channels that varies quadratically with the bending curvature. A model based on a mechanism analogous to the ovalization of hollow tubes enables us to predict quantitatively this nonlinear poroelastic response and identify the key physical parameters that control the generation of the pressure pulse. Further experiments conducted on natural tree branches reveal the same phenomenology. Once rescaled by the model prediction, both the biomimetic and natural branches fall on the same master curve, enlightening the universality of our poroelastic mechanism for the generation of hydraulic signals in plants.

Connecting the pieces: uncovering the molecular basis for long-distance communication through plant grafting.

Vascular plants are wired with a remarkable long-distance communication system. This network can span from as little as a few centimeters (or less) in species like Arabidopsis, up to 100 m in the tallest giant sequoia, linking distant organ systems into a unified, multicellular organism. Grafting is a fundamental technique that allows researchers to physically break apart and reassemble the long-distance transport system, enabling the discovery of molecular signals that underlie intraorganismal communication. In this review, we highlight how plant grafting has facilitated the discovery of new long-distance signaling molecules that function in coordinating developmental transitions, abiotic and biotic responses, and cross-species interactions. This rapidly expanding area of research offers sustainable approaches for improving plant performance in the laboratory, the field, the orchard, and beyond.

Damage to leaf veins suppresses root foraging precision.

PREMISE: Plants generally increase root growth in areas with high nutrients in heterogeneous soils, a phenomenon called foraging precision. The physiology of this process is not well understood, but it may involve shoot-root signaling via leaf veins. If this is true, then damage to leaf veins, but not to nearby mesophyll, would reduce plant foraging precision. METHODS: To test this hypothesis, we imposed two leaf damage treatments on Plantago asiatica and Prunus jamasakura, removing either the tip of each main vein or mesophyll tissue between the veins with a 3-mm-diameter hole punch. After 30 days or 20 weeks of plant growth, we measured root biomass in the soil in response to soil nutrient concentration. RESULTS: When leaf mesophyll was damaged, root biomass of both species was greater in nutrient-rich patches than in nutrient-poor patches. However, when leaf veins were damaged, root biomass was similar between patches. CONCLUSIONS: These results suggest the importance of shoot-root signaling in plants, emphasizing that physiological processes are not necessarily restricted to single organs. The idea that herbivores that damage leaf veins may affect a plant's ability to selectively forage in high-nutrient patches is novel, with implications for natural and managed systems.

Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots.

The OLIGOPEPTIDE TRANSPORTER 3 (OPT3) has recently been identified as a component of the systemic network mediating iron (Fe) deficiency responses in Arabidopsis. Reduced expression of OPT3 induces an over accumulation of Fe in roots and leaves, due in part by an elevated expression of the IRON-REGULATED TRANSPORTER 1. Here we show however, that opt3 leaves display a transcriptional program consistent with an Fe overload, suggesting that Fe excess is properly sensed in opt3 leaves and that the OPT3-mediated shoot-to-root signaling is critical to prevent a systemic Fe overload. We also took advantage of the tissue-specific localization of OPT3, together with other Fe-responsive genes, to determine the timing and location of early transcriptional events during Fe limitation and resupply. Our results show that the leaf vasculature responds more rapidly than roots to both Fe deprivation and resupply, suggesting that the leaf vasculature is within the first tissues that sense and respond to changes in Fe availability. Our data highlight the importance of the leaf vasculature in Fe homeostasis by sensing changes in apoplastic levels of Fe coming through the xylem and relaying this information back to roots via the phloem to regulate Fe uptake at the root level.