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SMALL AND MIGHTY: PEPTIDE HORMONES IN PLANT BIOLOGY
By Sonali Roy, Peter K. Lundquist, Michael K. Udvardi, and Wolf-Rüdiger Scheible What is a peptide hormone? 5-60 amino acid residues Serve as receptor ligands Influence many developmental programs Active at very low concentrations
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Small molecule hormones
Peptide hormones share many characteristics of classical plant hormones Small molecule hormones Peptide hormones Similarities Regulate growth and development even at minute concentrations Can act long distance Commonly require membrane receptors for recognition Activity may require secondary group modifications (e.g., hydroxylation) Chemical Structure- Derived from amino acids (e.g., Indole-3-acetic acid), isoprenoids (e.g., Benzyl amino purine), lipids (e.g., Methyl Jasmonate) Biosynthesis- End products of a cellular metabolic pathway Chemical Structure- Derived from polypeptide chains Biosynthesis- Transcribed ribosomally from a single gene SO4 Y I T N Phytosulfokine peptide Auxin (IAA)
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Peptide hormones control various developmental programs
Shoot development Meristem maintenance Cell division and expansion Plant height Biosynthesis of alkaloids Vascular differentiation Inflorescence development Inflorescence distribution Floral organ abscission Pollen tube germination, growth and guidance to ovule Stigma exudate deposition Self-incompatability determinant Fruit shape Leaf development Stomatal patterning and distribution Density of guard cells Leaf shape Leaf vascularization Seed and seed-pod development Early and late embryo patterning Nutrient translocation in seeds Response to biotic stresses Immune system and defense against pathogens Associations with beneficial microorganisms Protection against herbivory Root development Lateral root formation Meristem maintenance Gravitropism Root hair growth Casparian strip formation Vascular differentiation Response to abiotic stresses Nitrogen, phosphorus limitation Stomatal control of transpiration
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Peptide hormones can act over short and long distances
Cell-to-cell communication (Short distances) Organ-to-organ communication (Long distances) Example: In Legumes, members of the CLE family in roots signal to shoots when they have enough nitrogen-fixing organs! Example: Yellow cells indicate specific cells of the root tip in which the peptide CLE40 is expressed. SYMPLAST APOPLAST Image source: Root tip Modified from ?, Self
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Anatomy of a small signaling peptide
Two major criteria are used to classify plant peptides (1) Primary sequence characteristics (2) Post-translational modifications N-TERMINAL SIGNAL PEPTIDE VARIABLE REGION C-TERMINAL SIGNALLING PEPTIDE Directed to the endoplasmic reticulum, Golgi secretory pathway 5-60 amino acids long May contain different post-translational modifications Pre-pro-peptide Precursor of pro-peptide Biologically active peptide Inactive peptide
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Peptide hormones are classified into families
YES Does the precursor itself perform a function? YES NO Peptide has a different function Functional precursor Does the peptide have any post-translational modifications? YES FUNCTIONAL-PRECURSOR DERIVED PEPTIDES (Tyrosine) Can be sulphated Is the peptide derived from a single ribosomally translated precursor? NO (Proline) Can be hydroxylated, glycosylated POST-TRANSLATIONALLY MODIFIED PEPTIDES Does the peptide have four or more cysteines? YES Di-sulphide bridges form between cysteines NO CYSTEINE-RICH PEPTIDES 5’ region of mRNA Pri-miRNA Short ORF NONPRECURSOR DERIVED UNMODIFIED PEPTIDES NO See Tavormina, P., De Coninck, B., Nikonorova, N., De Smet, I., and Cammue, B.P.A. (2015). The Plant Peptidome: An expanding repertoire of structural features and biological functions. Plant Cell 27: 2095–2118.
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Peptide processing involves post-translational modifications
Can occur non-enzymatically – cysteine (C) disulfide bond formation This can be due to physio-chemical characteristics of reactive amino acids and the cellular environment (e.g. pH, oxygen etc.) May require chaperones. May require specific modifying enzymes (1) Prolyl 4-Hydroxylase These enzymes have highly tissue specific expression. Can be responsive to hypoxia, anoxia, mechanical wounding. (2) Tyrosylprotein sulfotransferase (TPST) TPST enzymes catalyze the transfer of sulfate to the phenolic group of the tyrosine residue. Absence of this enzyme results in many developmental defects including shorter roots.
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Three challenges to peptide hormone prediction
Bias against genome prediction of small genes SSP genes tend to be encoded in short gene products, however genome annotations under-predict or explicitly exclude short gene products, eliminating them from gene lists Only these longer ORFs are predicted to encode a gene Sequence conservation is limited to a few critical residues This family below has no conserved sequence pattern outside of the cysteines that can be used to search for additional genes Family Member 1 - Family Member 2 - Family Member 3 - Family Member 4 - * * * * * * * Rules for processing of a preproprotein are poorly defined Even when the genes are known it is difficult to determine what part of the gene product encodes the mature, bioactive peptide hormone Where does the peptidase cut? MKLQGITLNIINALFLMIFSFLLLSHSIQVEGTRPLKDQSAPSVISLIINQAYSGPSHKGRGH Full-length protein sequence Predicted Signal Peptide HKGRGH What is the resulting bioactive peptide? SGPSHKGRGH NQAYSGPSHKGRGH
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Discover Peptide Hormone Genes
The cooperative application of these techniques drives discovery of novel peptide hormones Transcriptomics Bioinformatics Discover Peptide Hormone Genes Advantage: Supports classification of an ORF as an active, expressed peptide hormone gene Limitation: Function and activity require confirmation from the other techniques Advantage: Can predict many SSP genes at a time Limitation: Must rely on prior knowledge & assumptions. Requires confirmation from the other techniques Biochemistry Genetics Advantage: Offers direct detection of a mature peptide and identification of underlying gene Limitation: Technically difficult to isolate and identify peptides Advantage: Connects peptide hormone genes to functions & phenotypes Limitation: Functional redundancy of genes can limit the potential of discovery
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Mechanisms of peptide hormone mobility
Peptide hormone expressed in one cell but receptor expressed in another cell So … how does a peptide move from its source to its receptor target? LOCAL LONG-DISTANCE Apoplastic diffusion Xylem stream Phloem translocation Shoot-expressed peptide hormones are loaded into the phloem Phloem-transported peptides can be distributed to the root tissue Murphy, E., Smith, S., & De Smet, I. (2012). Small signaling peptides in Arabidopsis development: How cells communicate over a short distance. Plant Cell 24: 3198–3217. Okamoto, S., Tabata, R., and Matsubayashi, Y. (2016). Long-distance peptide signaling essential for nutrient homeostasis in plants. Curr. Opin. Plant Biol. 34: 35–40. with permission.
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Methods to determine how a peptide is mobilized
Apoplast extraction Vascular sap extraction Root–shoot grafting Tests for: Presence in apoplastic fluid Tests for: Presence in vascular streams Tests for: Spatially-segregated expression of receptor/ligand Wild type Mutant Root peptide mutant Shoot receptor Apoplast: the extracellular space Sap is collected from the stem Vacuum infiltrate tissue with buffer Peptides are isolated from the xylem sap by liquid-liquid or solid-phase extraction Isolated peptides are identified by mass spectrometry Centrifuge tissue to extract apoplast/buffer mixture
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Receptor kinases recognize peptide hormones
Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) Ectodomain (Leucine-Rich Repeats) Perceive signal by binding peptide ligand Extracellular Matrix (apoplast) Transmembrane Segment Plasma Membrane Convey signal into the cell Cytosol Protein Kinase Initiate intracellular signaling
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Peptide hormones can be perceived by receptor/co-receptor pairs
Peptide receptors are typically Leucine-Rich Repeat Receptor-Like Kinases Receptor & co-receptor cooperate to bind the peptide ligand An IDA peptide in complex with its receptor & co-receptor (only the ectodomains are shown) Receptor (HAE) Peptide ligand (IDA) Co-receptor (SERK1) Santiago, J., Brandt, B., Wildhagen, M., Hohmann, U., Hothorn, L.A., Butenko, M.A., and Hothorn, M. (2016). Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission. eLife 5: e15075.
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Specific receptor/co-receptor combinations may specify the target ligand recognized
Peptide hormones, receptors & co-receptors are each in multi-gene families providing many possible combinations for interaction An example from the IDA Family Does the specific receptor/co-receptor pair determine which ligand to bind? The A. thaliana IDA family has six members IDA receptors & co-receptors are also in multi-gene families Peptide Hormone IDA IDA-LIKE 1 IDA-LIKE 2 IDA-LIKE 3 IDA-LIKE 4 IDA-LIKE 5 Receptor HAESA HAESA-LIKE 1 HAESA-LIKE 2 Receptor + co-receptor HAESA + SERK1 IDA HAESA-Like + SERK IDA-Like Co-receptor SERK1 SERK2 SERK3 SERK4 SERK5 Specificity for a ligand results in distinct downstream effects
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Signal attenuation is critical for proper peptide hormone function
Endocytosis Antagonism Sequestration Bioactive peptide Peptide pool Protein synthesis Vacuole (degradation) Antagonist Binds receptor Blocks binding of bioactive peptide Inactive Receptor Active Receptor Internalization positively regulates signaling Desensitizes cell by removing receptor & ligand E.g., triggered by binding of AtPEP1 or EF-TU Multiple ligands can compete for binding to a receptor Active ligand blocked from binding and activating the receptor E.g., EPFL9/EPF2 compete for binding to TMM receptor Inactive receptors bind and sequester a ligand Ligand prevented from binding and activating target receptor E.g., CLE40 is sequestered by ACR4
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CLE peptides regulate stem cell identity throughout the plant
The Arabidopsis Book Betsuyaku, S., Sawa, S., and Yamada, M. (2011). The Function of the CLE Peptides in Plant Development and Plant-Microbe Interactions. The Arabidopsis Book 9: e0149.
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IDA regulates cell separation events through cell wall remodeling & degradation
Floral organ abscission Abscission zone (PLANT BODY) (FLORAL ORGAN) Cell wall degradation in abscission zones Abscission zone IDA (ligand) HAE/HSL2 (receptor) Lateral root emergence Cell wall degradation in overlying cells Lateral root primordia Epidermis Emerging lateral root Cortex Endodermis Epidermis Pericycle Cortex Endodermis See Aalen, R.B. (2013). Maturing peptides open for communication. J. Exp. Bot. 64: 5231–5235. Image source: FigShare, Flickr
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Many peptide hormones are floral regulators
Peptide signaling in plant reproduction predominantly relies on the Cys-rich class Pollen grain Tapetum differentiation TPDs Pollen tube Self-incompatibility SCR Pollen germination PSKs, STIGs, LAT52/POE Pollen tube growth RALFs, STIGs, nsLTPs, PSKs Pollen tube attraction LUREs, EALs, PCYs, PSKs Egg cells Pollen tube burst ESs Egg–sperm fusion ECA1s
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Peptide hormones contribute to abiotic stress tolerance
TEMPERATURE IDA/IDAL PIP DROUGHT/DEHYDRATION CIF CLE Micronutrient deficiency/excess SULFUR, IRON NITROGEN CEP CLE Macronutrient deficiency PHOSPHORUS CLE
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Peptide hormones have roles in nitrogen deficiency responses
Changes in root system architecture Localized modifications of gene expression Interactions with beneficial soil rhizobia CEPD CEP Sufficient nitrogen Low nitrogen Vector control CEP1 overexpression clv1 mutants CLE2 overexpression WT Induction of nitrate uptake transporters NRT1.1 NRT2.1 NRT3.1 Induction of mobile CEPs Under low nitrogen conditions, clv1 receptor mutants produce excess lateral roots while overexpression of CLE2 peptides reduce LR number. Overexpression of CEP1 in Medicago enhances production of nodules that house nitrogen-fixing soil rhizobia. Araya, T., et al. (2014). CLE-CLAVATA1 peptide-receptor signaling module regulates the expansion of plant root systems in a nitrogen-dependent manner. Proc Natl Acad Sci U S A 111: Tabata, R., et al. (2014). Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling. Science 346: Reprinted with permission from AAAS Imin, N., et al. (2013). The peptide-encoding CEP1 gene modulates lateral root and nodule numbers in Medicago truncatula. J Exp Bot 64: by permission of Oxford University Press .
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Peptide hormones contribute to biotic stress resistance
Plant–bacterial interactions (pathogens) CAPE PEP GASA RALF Plant–herbivore interactions PIP CLE CAPE PEP GASA RALF SYSTEMIN Plant–nematode interactions CLE GASA Plant–bacterial interactions (symbionts) NCR CLE CEP PSK DVL1 Plant–fungal interactions PIP GASA PSK DEFENSINS
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Peptide hormones frequently appear in gene clusters, allowing neo-functionalization
Duplication of a stretch of the genome is caused by errors during genetic recombination Peptide hormone gene cluster Maintains original function and expression pattern Develops new expression pattern in different tissue type Develops binding affinity for different receptor Such gene duplication is thought to facilitate the rapid evolution seen among peptide-encoding genes
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Peptide hormones show high levels of species specificity
Examples of peptide hormone families that emerge at different points along the green plant lineage Many families develop within specific species or among closely-related species This indicates recent origin and is consistent with rapid evolution of peptide hormones CLE Family Vascular Plants Peptide Hormone Family Species GRIM REAPER (GRI) Arabidopsis thaliana Embryo Sac (ES) Maize (Zea mays) Nodule Cysteine Rich (NCR) Sub-clade of legumes LURE Various dicots ns-LTP Family Seed Plants IDA Family Flowering Plants RALF Family
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Peptide applications: Reality or wishful thinking?
Image Source: Pixabay Hormones as herbicides Peptides as growth/pest modulators ADVANTAGE Auxin-type herbicides (2,4-D or 2,4,5-T) selectively affect broadleaf plants and not cereals DISADVANTAGE Toxic if eaten or inhaled Persists in the environment for 1-14 days minimum ADVANTAGE Rapidly active at very low concentrations Completely biodegradable Easy to synthesize DISADVANTAGE Cross-reactivity with beneficial organisms?
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