The Raf-like MAPKKK INTEGRIN-LINKED KINASE 5 regulates purinergic receptor-mediated innate immunity in Arabidopsis.

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to an elevated plant defense response. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we show that in Arabidopsis thaliana, P2K1 phosphorylates the Raf-like MAP kinase kinase kinase (MAPKKK) INTEGRIN-LINKED KINASE 5 (ILK5) on serine 192 in the presence of eATP. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses. ILK5 interacts with and phosphorylates the MAP kinase MKK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type (WT). The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to WT plants. Plants expressing only the mutant ILK5S192A protein, with decreased kinase activity, did not activate the MAPK cascade upon ATP addition. These results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MPK3/6 associated with an elevated innate immune response.

The BADC and BCCP subunits of chloroplast acetyl-CoA carboxylase sense the pH changes of the light-dark cycle.

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a "fly-casting mechanism." We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light-dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity.

Label-Free Quantitative Phosphoproteomics Reveals Signaling Dynamics Involved in Embryogenic Competence Acquisition in Sugarcane.

In this study, a label-free quantitative phosphoproteomic analysis was performed to identify and quantify signaling events related to the acquisition of embryogenic competence in sugarcane. Embryogenic and nonembryogenic calli were compared at the multiplication phase, resulting in the identification of 163 phosphoproteins unique to embryogenic calli, 9 unique to nonembryogenic calli, and 51 upregulated and 40 downregulated in embryogenic calli compared to nonembryogenic calli. Data are available via ProteomeXchange with identifier PXD018054. Motif-x analysis revealed the enrichment of [xxxpSPxxx], [RxxpSxxx], and [xxxpSDxxx] motifs, which are predicted phosphorylation sites for several kinases related to stress responses. The embryogenic-related phosphoproteins (those unique and upregulated in embryogenic calli) identified in the present study are related to abscisic acid-induced signaling and abiotic stress response; they include OSK3, ABF1, LEAs, and RD29Bs. On the other hand, the nonembryogenic-related phosphoproteins EDR1 and PP2Ac-2 are negative regulators of abscisic acid signaling, suggesting a relationship between phosphoproteins involved in the abscisic acid and stress responses in the acquisition of embryogenic competence. Moreover, embryogenic-related phosphoproteins associated with epigenetic modifications, such as HDA6, HDA19, and TOPLESS, and with RNA metabolism, including AGO1, DEAH5, SCL30, UB2C, and SR45, were identified to play potential roles in embryogenic competence. These results reveal novel phosphorylation sites for several proteins and identify potential candidate biomarkers for the acquisition of embryogenic competence in sugarcane.

Multi-targeted trehalose-6-phosphate phosphatase I harbors a novel peroxisomal targeting signal 1 and is essential for flowering and development.

MAIN CONCLUSION: This work reveals information about new peroxisomal targeting signals type 1 and identifies trehalose-6-phosphate phosphatase I as multitargeted and is implicated in plant development, reproduction, and stress response. A putative, non-canonical peroxisomal targeting signal type 1 (PTS1) Pro-Arg-Met > was identified in the extreme C-terminus of trehalose-6-phosphate phosphatase (TPP)I. TPP catalyzes the final step of trehalose synthesis, and the enzyme was previously characterized to be nuclear only (Krasensky et al. in Antioxid Redox Signal 21(9):1289-1304, 2014). Here we show that the TPPI C-terminal decapeptide ending with Pro-Arg-Met > or Pro-Lys-Met > can indeed function as a PTS1. Upon transient expression in two plant expression systems, the free C- or N-terminal end led to the full-length TPPI targeting to peroxisomes and plastids, respectively. The nucleus and nucleolus targeting of the full-length TPPI was observed in both cases. The homozygous T-DNA insertion line of TPPI showed a pleiotropic phenotype including smaller leaves, shorter roots, delayed flowering, hypersensitivity to salt, and a sucrose dependent seedling development. Our results identify novel PTS1s, and TPPI as a protein multi-targeted to peroxisomes, plastids, nucleus, and nucleolus. Altogether our findings implicate an essential role for TPPI in development, reproduction, and cell signaling.

A Family of Negative Regulators Targets the Committed Step of de Novo Fatty Acid Biosynthesis.

Acetyl-CoA carboxylase (ACCase) catalyzes the committed step of de novo fatty acid biosynthesis. In prokaryotes, green algae, and most plants, this enzyme is a heteromeric complex requiring four different subunits for activity. The plant complex is recalcitrant to conventional purification schemes and hence the structure and composition of the full assembly have been unclear. In vivo coimmunoprecipitation using subunit-specific antibodies identified a novel family of proteins in Arabidopsis thaliana annotated as biotin/lipoyl attachment domain containing (BADC) proteins. Results from yeast two-hybrid and coexpression in Escherichia coli confirmed that all three BADC isoforms interact with the two biotin carboxyl carrier protein (BCCP) isoforms of Arabidopsis ACCase. These proteins resemble BCCP subunits but are not biotinylated due to a mutated biotinylation motif. We demonstrate that BADC proteins significantly inhibit ACCase activity in both E. coli and Arabidopsis. Targeted gene silencing of BADC isoform 1 in Arabidopsis significantly increased seed oil content when normalized to either mass or individual seed. We conclude the BADC proteins are ancestral BCCPs that gained a new function as negative regulators of ACCase after initial loss of the biotinylation motif. A functional model is proposed.

In Vivo Quantitative Monitoring of Subunit Stoichiometry for Metabolic Complexes.

Metabolic pathways often employ assemblies of individual enzymes to facilitate substrate channeling to improve thermodynamic efficiency and confer pathway directionality. It is often assumed that subunits to multienzyme complexes are coregulated and accumulate at fixed levels in vivo, reflecting complex stoichiometry. Such assumptions can be experimentally tested using modern tandem mass spectrometry, and herein we describe such an approach applied toward an important metabolic complex. The committed step of de novo fatty acid synthesis in the plastids of most plants is catalyzed by the multienzyme, heteromeric acetyl-CoA carboxylase (hetACCase). This complex is composed of four catalytic subunits and a recently discovered regulatory subunit resembling the biotin carboxyl carrier protein but lacking the biotinylation motif necessary for activity. To better understand this novel form of regulation, a targeted tandem mass-spectrometry-based assay was developed to absolutely quantify all subunits to the Arabidopsis thaliana hetACCase. After validation against pure, recombinant protein, this multiplexed assay was used to quantify hetACCase subunits in siliques in various stages of development. Quantitation provided a developmental profile of hetACCase and BADC protein expression that supports a recently proposed regulatory mechanism for hetACCase and demonstrates a promising application of targeted mass spectrometry for in vivo analysis of protein complexes.

Embryogenic Competence Acquisition in Sugar Cane Callus Is Associated with Differential H+-Pump Abundance and Activity.

Somatic embryogenesis is an important biological process in several plant species, including sugar cane. Proteomics approaches have shown that H+ pumps are differentially regulated during somatic embryogenesis; however, the relationship between H+ flux and embryogenic competence is still unclear. This work aimed to elucidate the association between extracellular H+ flux and somatic embryo maturation in sugar cane. We performed a microsomal proteomics analysis and analyzed changes in extracellular H+-flux and H+-pump (P-H+-ATPase, V-H+-ATPase, and H+-PPase) activity in embryogenic and non-embryogenic callus. A total of 657 proteins were identified, 16 of which were H+ pumps. We observed that P-H+-ATPase and H+-PPase were more abundant in embryogenic callus. Compared to non-embryogenic callus, embryogenic callus showed higher H+ influx, especially on maturation day 14, as well as higher H+-pump activity (mainly, P-H+-ATPase and H+-PPase activity). H+-PPase appears to be the major H+ pump in embryogenic callus during somatic embryo formation, functioning in both vacuole acidification and PPi homeostasis. These results provide evidence for an association between higher H+-pump protein abundance and, consequently, higher H+ flux and embryogenic competence acquisition in the callus of sugar cane, allowing for the optimization of the somatic embryo conversion process by modulating the activities of these H+ pumps.

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination.

Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLKs) are essential for plant growth, development, and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as coregulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as coregulatory and physical partners of EMS1. The SERK1/2 genes function in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation, Förster resonance energy transfer, and coimmunoprecipitation approaches revealed that SERK1 interacted biochemically with EMS1. Transphosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among 12 in vitro autophosphorylation and transphosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 autophosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence of the interaction and phosphorylation between SERK1/2 and EMS1 in anther development.

Proteomic Characterization of Bradyrhizobium diazoefficiens Bacteroids Reveals a Post-Symbiotic, Hemibiotrophic-Like Lifestyle of the Bacteria within Senescing Soybean Nodules.

The form and physiology of Bradyrhizobium diazoefficiens after the decline of symbiotic nitrogen fixation has been characterized. Proteomic analyses showed that post-symbiotic B. diazoefficiens underwent metabolic remodeling as well-defined groups of proteins declined, increased or remained unchanged from 56 to 119 days after planting, suggesting a transition to a hemibiotrophic-like lifestyle. Enzymatic analysis showed distinct patterns in both the cytoplasm and the periplasm. Similar to the bacteroid, the post-symbiotic bacteria rely on a non-citric acid cycle supply of succinate and, although viable, they did not demonstrate the ability to grow within the senescent nodule.

Luxurious Nitrogen Fertilization of Two Sugar Cane Genotypes Contrasting for Lignin Composition Causes Changes in the Stem Proteome Related to Carbon, Nitrogen, and Oxidant Metabolism but Does Not Alter Lignin Content.

Sugar cane is an important crop for sugar and biofuel production. Its lignocellulosic biomass represents a promising option as feedstock for second-generation ethanol production. Nitrogen fertilization can affect differently tissues and its biopolymers, including the cell-wall polysaccharides and lignin. Lignin content and composition are the most important factors associated with biomass recalcitrance to convert cell-wall polysaccharides into fermentable sugars. Thus it is important to understand the metabolic relationship between nitrogen fertilization and lignin in this feedstock. In this study, a large-scale proteomics approach based on GeLC-MS/MS was employed to identify and relatively quantify proteins differently accumulated in two contrasting genotypes for lignin composition after excessive nitrogen fertilization. From the ∼1000 nonredundant proteins identified, 28 and 177 were differentially accumulated in response to nitrogen from IACSP04-065 and IACSP04-627 lines, respectively. These proteins were associated with several functional categories, including carbon metabolism, amino acid metabolism, protein turnover, and oxidative stress. Although nitrogen fertilization has not changed lignin content, phenolic acids and lignin composition were changed in both species but not in the same way. Sucrose and reducing sugars increased in plants of the genotype IACSP04-065 receiving nitrogen.

Regulation and structure of the heteromeric acetyl-CoA carboxylase.

The enzyme acetyl-CoA carboxylase (ACCase) catalyzes the committed step of the de novo fatty acid biosynthesis (FAS) pathway by converting acetyl-CoA to malonyl-CoA. Two forms of ACCase exist in nature, a homomeric and heteromic form. The heteromeric form of this enzyme requires four different subunits for activity: biotin carboxylase; biotin carboxyl carrier protein; and α- and ß-carboxyltransferases. Heteromeric ACCases (htACCase) can be found in prokaryotes and the plastids of most plants. The plant htACCase is regulated by diverse mechanisms reflected by the biochemical and genetic complexity of this multienzyme complex and the plastid stroma where it resides. In this review we summarize the regulation of the plant htACCase and also describe the structural characteristics of this complex from both prokaryotes and plants. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

The Bacteroid Periplasm in Soybean Nodules Is an Interkingdom Symbiotic Space.

The functional role of the periplasm of nitrogen-fixing bacteroids has not been determined. Proteins were isolated from the periplasm and cytoplasm of Bradyrhizobium diazoefficiens bacteroids and were analyzed using liquid chromatography tandem mass spectrometry proteomics. Identification of bacteroid periplasmic proteins was aided by periplasm prediction programs. Approximately 40% of all the proteins identified as periplasmic in the B. diazoefficiens genome were found expressed in the bacteroid form of the bacteria, indicating the periplasm is a metabolically active symbiotic space. The bacteroid periplasm possesses many fatty acid metabolic enzymes, which was in contrast to the bacteroid cytoplasm. Amino acid analysis of the periplasm revealed an abundance of phosphoserine, phosphoethanolamine, and glycine, which are metabolites of phospholipid metabolism. These results suggest the periplasm is a unique space and not a continuum with the peribacteroid space. A number of plant proteins were found in the periplasm fraction, which suggested contamination. However, antibodies to two of the identified plant proteins, histone H2A and lipoxygenase, yielded immunogold labeling that demonstrated the plant proteins were specifically targeted to the bacteroids. This suggests that the periplasm is an interkingdom symbiotic space containing proteins from both the bacteroid and the plant.

The Raf-like Kinase ILK1 and the High Affinity K+ Transporter HAK5 Are Required for Innate Immunity and Abiotic Stress Response.

Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca(2+) uptake in signaling, the regulation and significance of PAMP-induced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H(+))/K(+) symporter that mediates a high-affinity uptake during K(+) deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K(+) efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K(+) loss. Taken together, our results position ILK1 as a link between plant defense pathways and K(+) homeostasis.

A laser ablation ICP-MS based method for multiplexed immunoblot analysis: applications to manganese-dependent protein dynamics of photosystem II in barley (Hordeum vulgare L.).

Manganese (Mn) constitutes an essential co-factor in the oxygen-evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we developed a multiplexed antibody-based assay and analysed selected PSII subunits in barley (Hordeum vulgare L.). A selection of antibodies were labelled with specific lanthanides and immunoreacted with thylakoids exposed to Mn deficiency after western blotting. Subsequently, western blot membranes were analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods, based on data-dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re-supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn-dependent changes in abundances. Similar trends in the response to Mn deficiency at the protein level were observed when comparing DDA, SRM and LA-ICP-MS results. A biologically important exception to this trend was the loss of PsbO in the SRM analysis, which highlights the necessity of validating protein changes by more than one technique. The developed method enables a higher number of proteins to be multiplexed in comparison to existing immunoassays. Furthermore, multiplexed protein analysis by LA-ICP-MS provides an analytical platform with high throughput appropriate for screening large collections of plants.

ACYL-LIPID DESATURASE2 is required for chilling and freezing tolerance in Arabidopsis.

Fatty acid desaturation of membrane lipids is a strategy for plants to survive chilling or freezing temperature. To further characterize enzymes involved in this stress response pathway, ACYL-LIPID DESATURASE2 (ADS2; Enzyme Commission 1.14.99) was studied using genetic, cell, and biochemical approaches. ads2 mutant plants appear similar to the wild type under standard growth conditions but display a dwarf and sterile phenotype when grown at 6°C and also show increased sensitivity to freezing temperature. Fatty acid composition analysis demonstrated that ads2 mutant plants at 6°C have reduced levels of 16:1, 16:2, 16:3, and 18:3 and higher levels of 16:0 and 18:0 fatty acids compared with the wild type. Lipid profiling revealed that 34C species of phosphatidylglycerol (PG) and monogalactosyl diacylglycerol (MGDG) content in ads2 mutants were lower and phosphatidic acid, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, lyso-phosphatidylcholine, and phosphatidylserine were higher than the wild type. Subcellular localization of C- and N-terminal enhanced fluorescence fusion proteins indicated that ADS2 localized primarily to the endoplasmic reticulum, although signal was also confirmed in Golgi and plastids. A double mutation with a putative plastid ADS3 paralog exacerbates the growth defects of ads2 mutant plants under low temperature. These observations suggest that ADS2 encodes a 16:0 desaturase of MGDG and PG. We hypothesize that a low temperature-induced shift from the plastid to endoplasmic reticulum pathway for membrane lipid biosynthesis is required for the cold stress response in Arabidopsis thaliana, and ADS2 is essential to adjust the acyl composition of organelle membrane lipid composition in response to cold stress.

Acyl-lipid desaturase 1 primes cold acclimation response in Arabidopsis.

Membrane fluidity change has long been suggested as the primary mechanism by which, plants adapt to cold stress, but the underlying molecular mechanisms are not completely established. In this study, we found that a knockout of acyl-lipid/CoA desaturase 1 gene (ADS1; EC 1.14.99) enhances freezing tolerance after cold acclimation (CA). Fatty acid composition analysis demonstrated that 18:1 content in ads1 mutant plants was 20% lower than in wild-type (WT) grown at 23°C. Lipidomics revealed that 34C-species of monogalactosyl diacylglycerol (MGDG) content in ads1 mutants were 3.3-14.9% lower than in WT. Lipid positional analysis identified 10% lower 18:1 fatty acid content at the sn-2 position of MGDG. The cytosolic calcium content in ads1 mutant plants was also approximately two-times higher than that of WT in response to cold shock. Each of these biochemical differences between WT and ads1 mutant disappeared after CA. Subcellular localization of C- and N-terminal enhanced-fluorescence-fusion proteins indicated that ADS1 localized exclusively to chloroplasts. These observations suggest that ADS1-mediated alteration of chloroplast membrane fluidity is required to prime a CA response, and is the upstream event of cytosolic calcium signaling.

Genome sequence of the palaeopolyploid soybean.

Soybean (Glycine max) is one of the most important crop plants for seed protein and oil content, and for its capacity to fix atmospheric nitrogen through symbioses with soil-borne microorganisms. We sequenced the 1.1-gigabase genome by a whole-genome shotgun approach and integrated it with physical and high-density genetic maps to create a chromosome-scale draft sequence assembly. We predict 46,430 protein-coding genes, 70% more than Arabidopsis and similar to the poplar genome which, like soybean, is an ancient polyploid (palaeopolyploid). About 78% of the predicted genes occur in chromosome ends, which comprise less than one-half of the genome but account for nearly all of the genetic recombination. Genome duplications occurred at approximately 59 and 13 million years ago, resulting in a highly duplicated genome with nearly 75% of the genes present in multiple copies. The two duplication events were followed by gene diversification and loss, and numerous chromosome rearrangements. An accurate soybean genome sequence will facilitate the identification of the genetic basis of many soybean traits, and accelerate the creation of improved soybean varieties.

P³DB 3.0: From plant phosphorylation sites to protein networks.

In the past few years, the Plant Protein Phosphorylation Database (P(3)DB, http://p3db.org) has become one of the most significant in vivo data resources for studying plant phosphoproteomics. We have substantially updated P(3)DB with respect to format, new datasets and analytic tools. In the P(3)DB 3.0, there are altogether 47 923 phosphosites in 16 477 phosphoproteins curated across nine plant organisms from 32 studies, which have met our multiple quality standards for acquisition of in vivo phosphorylation site data. Centralized by these phosphorylation data, multiple related data and annotations are provided, including protein-protein interaction (PPI), gene ontology, protein tertiary structures, orthologous sequences, kinase/phosphatase classification and Kinase Client Assay (KiC Assay) data--all of which provides context for the phosphorylation event. In addition, P(3)DB 3.0 incorporates multiple network viewers for the above features, such as PPI network, kinase-substrate network, phosphatase-substrate network, and domain co-occurrence network to help study phosphorylation from a systems point of view. Furthermore, the new P(3)DB reflects a community-based design through which users can share datasets and automate data depository processes for publication purposes. Each of these new features supports the goal of making P(3)DB a comprehensive, systematic and interactive platform for phosphoproteomics research.

Comparative proteomic analysis of developing rhizomes of the ancient vascular plant Equisetum hyemale and different monocot species.

The rhizome is responsible for the invasiveness and competitiveness of many plants with great economic and agricultural impact worldwide. Besides its value as an invasive organ, the rhizome plays a role in the establishment and massive growth of forage, providing biomass for biofuel production. Despite these features, little is known about the molecular mechanisms that contribute to rhizome growth, development, and function in plants. In this work, we characterized the proteome of rhizome apical tips and elongation zones from different species using a GeLC-MS/MS (one-dimensional electrophoresis in combination with liquid chromatography coupled online with tandem mass spectrometry) spectral-counting proteomics strategy. Five rhizomatous grasses and an ancient species were compared to study the protein regulation in rhizomes. An average of 2200 rhizome proteins per species were confidently identified and quantified. Rhizome-characteristic proteins showed similar functional distributions across all species analyzed. The over-representation of proteins associated with central roles in cellular, metabolic, and developmental processes indicated accelerated metabolism in growing rhizomes. Moreover, 61 rhizome-characteristic proteins appeared to be regulated similarly among analyzed plants. In addition, 36 showed conserved regulation between rhizome apical tips and elongation zones across species. These proteins were preferentially expressed in rhizome tissues regardless of the species analyzed, making them interesting candidates for more detailed investigative studies about their roles in rhizome development.