Function and solution structure of the Arabidopsis thaliana RALF8 peptide.

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine-rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well-resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N-terminal His-tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with 15 N and 13 C for NMR analysis and obtained near complete 1 H, 13 C, and 15 N NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges.

Probing a Plant Plasma Membrane Receptor Kinase's Three-Dimensional Structure Using Mass Spectrometry-Based Protein Footprinting.

FERONIA (FER), one of the 17 malectin-like receptor-like kinases encoded in the Arabidopsis genome, acts as a receptor for a 5 kDa growth-inhibiting secreted protein hormone, rapid alkalinization factor 1 (RALF1). Upon binding the peptide ligand, FER is involved in a variety of signaling pathways eliciting ovule fertilization and vegetative root cell expansion. Here, we report the use of mass spectrometry-based, carbodiimide-mediated protein carboxyl group (aspartic and glutamic acid) footprinting to map solvent accessible amino acids of the ectodomain of FER (ectoFER), including those involved in RALF1 binding and/or allosteric changes. Aspartate and glutamate residues labeled in this procedure were located in various regions, including the N-terminus, malectin-like domains, and juxtamembrane region, and these correlated well with a three-dimensional structural model of ectoFER predicted from the crystal structure of a related receptor. Covalent cross-linking experiments also revealed the N-terminus of ectoFER linked to the highly conserved C-terminus of RALF1. RALF1 binding assays performed with truncation mutants of ectoFER further implicated the receptor N-terminal and juxtamembrane regions in the binding of RALF1. In conclusion, our results of mass spectrometry-based footprinting methods provide a framework for understanding ligand-induced changes in solvent accessibility and their positions within the three-dimensional structure of a plant receptor kinase.

Comparison of the effects of a kinase-dead mutation of FERONIA on ovule fertilization and root growth of Arabidopsis.

A plasma membrane receptor protein kinase, FERONIA (FER), regulates various aspects of plant reproductive and vegetative growth. In roots, binding of a peptide ligand to FER causes rapid suppression of cell elongation whereas in ovules, FER is involved in gametophyte interactions. Here, we examined the effect of a mutation that eliminates kinase activity, on both ovule fertilization and root growth, using the same batch of seeds containing a kinase-dead mutation. The kinase-dead mutation of FER reduced the ability to complement fer-4 knockout phenotypes, compared with wild-type sequence in root, but not in ovules. Our results support a model in which cell type-specific regulatory mechanisms, such as different interacting partners and/or downstream signaling events, lead to cell type-specific functions of FER.

Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase.

A P-type H+-ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis (Arabidopsis thaliana) plant expressing H+-ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H+ secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H+-ATPase.

A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

There are more than 600 receptor-like kinases (RLKs) in Arabidopsis, but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins.

Ligand Receptor-Mediated Regulation of Growth in Plants.

Growth and development of multicellular organisms are coordinately regulated by various signaling pathways involving the communication of inter- and intracellular components. To form the appropriate body patterns, cellular growth and development are modulated by either stimulating or inhibiting these pathways. Hormones and second messengers help to mediate the initiation and/or interaction of the various signaling pathways in all complex multicellular eukaryotes. In plants, hormones include small organic molecules, as well as larger peptides and small proteins, which, as in animals, act as ligands and interact with receptor proteins to trigger rapid biochemical changes and induce the intracellular transcriptional and long-term physiological responses. During the past two decades, the availability of genetic and genomic resources in the model plant species, Arabidopsis thaliana, has greatly helped in the discovery of plant hormone receptors and the components of signal transduction pathways and mechanisms used by these immobile but highly complex organisms. Recently, it has been shown that two of the most important plant hormones, auxin and abscisic acid (ABA), act through signaling pathways that have not yet been recognized in animals. For example, auxins stimulate cell elongation by bringing negatively acting transcriptional repressor proteins to the proteasome to be degraded, thus unleashing the gene expression program required for increasing cell size. The "dormancy" inducing hormone, ABA, binds to soluble receptor proteins and inhibits a specific class of protein phosphatases (PP2C), which activates phosphorylation signaling leading to transcriptional changes needed for the desiccation of the seeds prior to entering dormancy. While these two hormone receptors have no known animal counterparts, there are also many similarities between animal and plant signaling pathways. For example, in plants, the largest single gene family in the genome is the protein kinase family (approximately 5% of the protein coding genes), although the specific function for only a few dozen of these kinases is clearly established. Recent comparative genomics studies have revealed that parasitic nematodes and pathogenic microbes produce plant peptide hormone mimics that target specific plant plasma membrane receptor-like protein kinases, thus usurping endogenous signaling pathways for their own pathogenic purposes. With biochemical, genetic, and physiological analyses of the regulation of hormone receptor signal pathways, we are thus just now beginning to understand how plants optimize the development of their body shape and cope with constantly changing environmental conditions.

Regulation of the plasma membrane proton pump (H(+)-ATPase) by phosphorylation.

In plants and fungi, energetics at the plasma membrane is provided by a large protonmotive force (PMF) generated by the family of P-type ATPases specialized for proton transport (commonly called PM H(+)-ATPases or, in Arabidopsis, AHAs for Arabidopsis H(+)-ATPases). Studies have demonstrated that this 100-kDa protein is essential for plant growth and development. Posttranslational modifications of the H(+)-ATPase play crucial roles in its regulation. Phosphorylation of several Thr and Ser residues within the carboxy terminal regulatory domain composed of ∼100 amino acids change in response to environmental stimuli, endogenous hormones, and nutrient conditions. Recently developed mass spectrometric technologies provide a means to carefully quantify these changes in H(+)-ATPase phosphorylation at the different sites. These chemical modifications can then be genetically tested in planta by complementing the loss-of-function aha mutants with phosphomimetic mutations. Interestingly, recent data suggest that phosphatase-mediated changes in PM H(+)-ATPase phosphorylation are important in mediating auxin-regulated growth. Thus, as with another hormone (abscisic acid), dephosphorylation by phosphatases, rather than kinase mediated phosphorylation, may be an important focal point for regulation during plant signal transduction. Although interactions with other proteins have also been implicated in ATPase regulation, the very hydrophobic nature and high concentration of this polytopic protein presents special challenges in evaluating the biological significance of these interactions. Only by combining biochemical and genetic experiments can we attempt to meet these challenges to understand the essential molecular details by which this protein functions in planta.

Functional characterization of PCRK1, a putative protein kinase with a role in immunity.

In Arabidopsis, defense signaling is triggered by the perception of conserved molecular patterns by pattern recognition receptors (PRRs). Signal transduction from the PRRs requires members of a family of Receptor-Like Cytoplasmic Kinases (RLCKs). Previously, we described one such RLCK, PTI Compromised Receptor-Like Cytoplasmic Kinase 1 (PCRK1) that is important for immunity induced by Microbe Associated Molecular Patterns (MAMPs) as well as Damage Associated Molecular Patterns (DAMPs). In this study, we measured the growth of Pma ES4326 in double mutants carrying pcrk1 together with the salicylic acid (SA) biosynthesis mutation sid2-2 or the jasmonic acid (JA) receptor mutation coi1-1, showing that the function of PCRK1 is SA independent but may be partially dependent on JA. Mutation of phosphorylated serine residues S232, S233 and S237 compromised the immune signaling function of PCRK1.

A peptide hormone and its receptor protein kinase regulate plant cell expansion.

Plant cells are immobile; thus, plant growth and development depend on cell expansion rather than cell migration. The molecular mechanism by which the plasma membrane initiates changes in the cell expansion rate remains elusive. We found that a secreted peptide, RALF (rapid alkalinization factor), suppresses cell elongation of the primary root by activating the cell surface receptor FERONIA in Arabidopsis thaliana. A direct peptide-receptor interaction is supported by specific binding of RALF to FERONIA and reduced binding and insensitivity to RALF-induced growth inhibition in feronia mutants. Phosphoproteome measurements demonstrate that the RALF-FERONIA interaction causes phosphorylation of plasma membrane H(+)-adenosine triphosphatase 2 at Ser(899), mediating the inhibition of proton transport. The results reveal a molecular mechanism for RALF-induced extracellular alkalinization and a signaling pathway that regulates cell expansion.

The effect of a genetically reduced plasma membrane protonmotive force on vegetative growth of Arabidopsis.

The plasma membrane proton gradient is an essential feature of plant cells. In Arabidopsis (Arabidopsis thaliana), this gradient is generated by the plasma membrane proton pump encoded by a family of 11 genes (abbreviated as AHA, for Arabidopsis H(+)-ATPase), of which AHA1 and AHA2 are the two most predominantly expressed in seedlings and adult plants. Although double knockdown mutant plants containing T-DNA insertions in both genes are embryonic lethal, under ideal laboratory growth conditions, single knockdown mutant plants with a 50% reduction in proton pump concentration complete their life cycle without any observable growth alteration. However, when grown under conditions that induce stress on the plasma membrane protonmotive force (PMF), such as high external potassium to reduce the electrical gradient or high external pH to reduce the proton chemical gradient, aha2 mutant plants show a growth retardation compared with wild-type plants. In this report, we describe the results of studies that examine in greater detail AHA2's specific role in maintaining the PMF during seedling growth. By comparing the wild type and aha2 mutants, we have measured the effects of a reduced PMF on root and hypocotyl growth, ATP-induced skewed root growth, and rapid cytoplasmic calcium spiking. In addition, genome-wide gene expression profiling revealed the up-regulation of potassium transporters in aha2 mutants, indicating, as predicted, a close link between the PMF and potassium uptake at the plasma membrane. Overall, this characterization of aha2 mutants provides an experimental and theoretical framework for investigating growth and signaling processes that are mediated by PMF-coupled energetics at the cell membrane.

Molecular characterization of mutant Arabidopsis plants with reduced plasma membrane proton pump activity.

Arabidopsis mutants containing gene disruptions in AHA1 and AHA2, the two most highly expressed isoforms of the Arabidopsis plasma membrane H(+)-ATPase family, have been isolated and characterized. Plants containing homozygous loss-of-function mutations in either gene grew normally under laboratory conditions. Transcriptome and mass spectrometric measurements demonstrate that lack of lethality in the single gene mutations is not associated with compensation by increases in RNA or protein levels. Selected reaction monitoring using synthetic heavy isotope-labeled C-terminal tryptic peptides as spiked standards with a triple quadrupole mass spectrometer revealed increased levels of phosphorylation of a regulatory threonine residue in both isoforms in the mutants. Using an extracellular pH assay as a measure of in vivo ATPase activity in roots, less proton secreting activity was found in the aha2 mutant. Among 100 different growth conditions, those that decrease the membrane potential (high external potassium) or pH gradient (high external pH) caused a reduction in growth of the aha2 mutant compared with wild type. Despite the normal appearance of single mutants under ideal laboratory growth conditions, embryos containing homozygous double mutations are lethal, demonstrating that, as expected, this protein is absolutely essential for plant cell function. In conclusion, our results demonstrate that the two genes together perform an essential function and that the effects of their single mutations are mostly masked by overlapping patterns of expression and redundant function as well as by compensation at the post-translational level.

The Phaeodactylum genome reveals the evolutionary history of diatom genomes.

Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one-fifth of the primary productivity on Earth. The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology. Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes ( approximately 40%) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans.

A cytoplasmic Ca2+ functional assay for identifying and purifying endogenous cell signaling peptides in Arabidopsis seedlings: identification of AtRALF1 peptide.

Transient increases in the cytoplasmic Ca(2+) concentration are key events that initiate many cellular signaling pathways in response to developmental and environmental cues in plants; however, only a few extracellular mediators regulating cytoplasmic Ca(2+) singling are known to date. To identify endogenous cell signaling peptides regulating cytoplasmic Ca(2+) signaling, Arabidopsis seedlings expressing aequorin were used for an in vivo luminescence assay for Ca(2+) changes. These seedlings were challenged with fractions derived from plant extracts. Multiple heat-stable, protease-sensitive peaks of calcium elevating activity were observed after fractionation of these extracts by high-performance liquid chromatography. Tandem mass spectrometry identified the predominant active molecule isolated by a series of such chromatographic separations as a 49-amino acid polypeptide, AtRALF1 (the rapid alkalinization factor protein family). Within 40 s of treatment with nanomolar concentrations of the natural or synthetic version of the peptides, the cytoplasmic Ca(2+) level increased and reached its maximum. Prior treatment with a Ca(2+) chelator or inhibitor of IP 3-dependent signaling partially suppressed the AtRALF1-induced Ca(2+) concentration increase, indicating the likely involvement of Ca(2+) influx across the plasma membrane as well as release of Ca(2+) from intracellular reserves. Ca(2+) imaging using seedlings expressing the FRET-based Ca(2+) sensor yellow cameleon (YC) 3.6 showed that AtRALF1 could induce an elevation in Ca(2+) concentration in the surface cells of the root consistent with the very rapid effects of addition of AtRALF1 on Ca(2+) levels as reported by aequorin. Our data support a model in which the RALF peptide mediates Ca(2+)-dependent signaling events through a cell surface receptor, where it may play a role in eliciting events linked to stress responses or the modulation of growth.

Rapid alkalinization factors in poplar cell cultures. Peptide isolation, cDNA cloning, and differential expression in leaves and methyl jasmonate-treated cells.

A family of peptides inducing rapid pH alkalinization in hybrid poplar (Populus trichocarpa x Populus deltoides) cell culture medium was isolated from hybrid poplar leaves. Five related approximately 5-kD peptides were purified by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption ionization-mass spectrometry. The N-terminal sequence of one of the isolated peptides was very similar to a previously characterized peptide from tobacco (Nicotiana tabacum), rapid alkalinization factor (RALF), which causes a rapid increase in culture medium pH when added to tobacco cell cultures (G. Pearce, D.S. Moura, J. Stratmann, C.A. Ryan [2001] Proc Natl Acad Sci USA 98: 12843-12847). Two unique poplar RALF cDNAs (PtdRALF1 and PtdRALF2) were isolated from a poplar cDNA library and used to study RALF expression in poplar saplings and cultured poplar cells. Both genes were found to be expressed constitutively in poplar saplings and cultured cells. However, PtdRALF2 was expressed in leaves at very low levels, and its expression in suspension culture cells was transiently suppressed by methyl jasmonate (MeJa). Although the function of these novel peptides remains enigmatic, our experiments suggest their role may be developmental rather than stress related. Overall, our study confirms the presence of active RALF peptides in other plants, and provides new data on the complexity of the RALF gene family in poplar.

Polyphenol oxidase and herbivore defense in trembling aspen (Populus tremuloides): cDNA cloning, expression, and potential substrates.

The biochemical anti-herbivore defense of trembling aspen (Populus tremuloides Michx.) was investigated in a molecular analysis of polyphenol oxidase (PPO; EC 1.10.3.2). A PPO cDNA was isolated from a trembling aspen wounded leaf cDNA library and its nucleotide sequence determined. Southern analysis indicated the presence of two PPO genes in the trembling aspen genome. Expression of PPO was found to be induced after herbivory by forest tent caterpillar, by wounding, and by methyl jasmonate treatment. Wound induction was systemic, and occurred in unwounded leaves on wounded plants. This pattern of expression is consistent with a role of this enzyme in insect defense. A search for potential PPO substrates in ethanolic aspen leaf extracts using electron spin resonance (ESR) found no pre-existing diphenolic compounds. However, following a brief delay and several additions of oxygen, an ESR signal specific for catechol was detected. The source of this catechol was most likely the aspen phenolic glycosides tremulacin or salicortin which decomposed during ESR experiments. This was subsequently confirmed in experiments using pure salicortin.