For many years the classical plant hormones have studied as key regulators of plant growth and development, but in the past few years, research indicate that peptide are also play an important role in plant signaling for plant growth and developmental processes such as defense responses, cell elongation, cell proliferation and differentiation, meristem organization, nodule development, self incompatibility and organ abscission etc. In plants peptides are synthesized by using mRNA as a template and most often go to post translational modifications to yield mature peptide. Here in this review paper we are trying to provide an overview on peptide hormones and their functions in plant growth and development.
Signal transduction is very important for the development of multicellular organisms (whether they are animal or plant) and also so for the function of various organ systems. In plants cell signaling is mainly mediated by small lipophilic compounds (so called as classical plant hormones) such as auxins, gibberellins, cytokinins, ethylene and abscissic acid1. Later on, some other compounds such as brassinosteroids, jasmonates, salicylates, strigolactones and karrikins have also been added to the list of plant growth regulators2-3. Research findings of over the last few decades indicate that beside these plant hormones other molecules, including peptide hormones (also known as signaling or secreted peptides), small RNAs and transcription factors are also play an important role in signaling2,4-5. Peptide hormones are now widely accepted as signaling messengers in plants for their involvement in various aspects of plant growth regulation including defense responses, callus growth, meristem organization, self incompatibility, root growth, leaf shape regulation, nodule development and organ abscission etc.1. Most of these peptides have been identified by biochemical purification and genetic studies.
Peptide hormones often produced as larger molecular weight precursors that are proteolytically cleaved to the active form of the hormone and are water soluble. Plant peptides are active in the nanomolar to picomolar range6-7. Although the first peptide signal in plants was reported in 1991, little documentation is available for peptide signaling in higher plants compared with the documentation available for signaling in animals. In this review paper some peptide hormones are discussed with their importance in biological processes of plant development and growth.
On the basis of structural characteristics the plant peptide hormones are classified into two groups in which first group is consists of cysteine rich peptides and the second group consists of cysteine poor peptides also known as small post translationally modified peptides. Like other proteins, peptide hormones are also synthesized by using mRNA as a template; their precursors are known to be processed either in the endoplasmic reticulum or outside the cell. They are also sometimes modified by the processes such as glycosylation and hydroxylation8. The secreted peptide encoding genes are transcribed first and after that they are translated as pre-propeptides and then signal peptidase remove the N-terminal signal peptidase to produce propeptides. The produced propeptides are further modified by several enzymes, yielding functional mature peptides9. However based on their biosynthetic pathway the signaling peptides are classified into three different groups i.e. small post translationally modified peptides, cysteine rich peptides and intermediate type peptides5,10
The first group of peptide hormones is of the small post translationally modified peptides, consists of ?20 amino acids. The members of this group are cysteine poor peptides. The propeptides corresponding to the mature peptides consists of approximately 70-120 amino acids. These are always resulting from proteolytic processing and have a C-terminal conserved motif that often carries proline residues and post translational modifications9,11. Most of the plant hormones of plants are belonging to this group such as PSK, CLV3/ESR, PSY1, CEP and RGF/GLV/CLEL12-22. The second group of the peptide hormones has cysteine rich peptides. These are characterized by the presence of an even (usually six or eight) number of cysteine residues. The three dimensional structure of the mature protein is determined by the interamolecular disulfide bonds. The peptide belonging to this group may be or may not be has proteolytic processing. The mature peptides of this group are usually having large size usually more than 20 amino acids9,11. The cysteine rich peptides include the SCR/SP11 and LUREs23-25. Another group of the peptide hormones is referred as the intermediate type peptides, is intermediate between above mentioned both groups i.e. small post-translationally modified peptides and the cysteine rich peptides. Although intermediate type peptides have intramolecular disulfide bonds like cysteine rich peptide, these peptides are also produced via proteolytic processing similar to the small post translationally modified peptides9. In these peptides, the cysteine residues are located within the C-terminal region of the propeptides. Stomagens and RALFs are the representative of this intermediate-type peptide group26-28.
In plants, the maturation of small post translationally modified peptides is occurred through the involving of three types of post translation modification processes i.e. tyrosine sulfation, proline hydroxylation and hydroxyproline arabinosylation.
Tyrosine sulfation is catalyzed by tyrosyl protein sulfotransferase, a golgibody-localized enzyme which catalyzes the transfer of a sulfonate moiety from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to the hydroxyl group of a protein bound tyrosine residue29. Tyrosine sulfation occurs widely in multicellular organisms and modulates biological activity of proteins, the proteolytic processing of bioactive peptides, and extracellular protein-protein interactions10,30. PSK, PSYI and RGF1 are the identified tyrosine sulfated peptides in plants16,20,31.
Proline hydroxylation is catalyzed by the prolyl-4-hydoxylase (P4H), which is localized in both the endoplasmic reticulum and golgi apparatus. P4H is a protein with N-terminal transmembrane domain and is a member of a family of 2-oxoglutarate-dependent dioxygenases32. Proline hydroxylated peptides have been identified in plants are TDIF, CLV3, PSY1 and RGF113,16,17,20,33.
Hydroxyproline arabinosylation is catalyzed by hydroxyproline O-arabinosyltransferase (HPAT) which catalyzes the transfer of L-arabinose to the hydroxyl group of hydroxyproline residues34. HAPT is a golgi apparatus-localized transmembrane protein that is structurally similar to members of the glycosyltransferase GT8 family. Of the three HPAT genes present in the Arabidopsis thaliana genome, HPAT3 plays the central role in the arabinosylation of CLE peptides9,34.
Many higher plants respond to wounds from insect attack by producing defense proteins in leaves and stems35. In the leaves of solanaceous plants like tomato and potato, the defense proteins (i.e. protease inhibitor I and II) have been detected which interfere with the protein digestion of attacking insect and retard their growth and development36. It is found that these defense proteins accumulate not only in wounded leaves but also in the undamaged leaves far from the damage sites. This suggests that this systemic defense response is induced by a long distance signal transmission. The factor involve in this long distance signaling was isolated from the tomato leaves and named as systemin (TomSys due to isolated from tomato)6. Systemin consists of 18 amino acids and was the first isolated peptide hormones in plants. However several studies suggest that wound induced release of TomSys into the vascular system activates jasmonic acid biosynthesis in surrounding vascular tissues, and that the resulting jasmonic acid acts as a long-distance signal including a systemic wound response37.
The precursor to tomato systemin is transcribed as a 200 amino acid polypeptide38. It does not contain a putative signal sequence suggesting that it is synthesized on ribosomes in the cytosol39. In unwounded tomato leaves mRNA encoding systemin precursor is present in very small amounts but accumulates upon wounding mainly in the sieve elements surrounding cells of the phloem tissues in vascular bundles of mid veins. After wounding the systemin precursor stored solely in the phloem parenchyma cells of tomato leaves.
Hormonal, environmental or pathogenic signals are mostly perceived by membrane localized receptors that transduce those signals inside plant cells to activate programs directing growth, development, and defense responses. In the fully sequenced Arabidopsis genome, there are more than 600 receptor like kinase (RLK) genes about 200 of which belongs to a family called leucine-rich repeat (LRR) receptor-like kinases. Despite of the large number of LRR-RLKs in plants, fewer than 10 of them have known biological functions such as hormone perception, meristem signaling and pathogen responses40. A tomato LRR-RLK known as SR160 has been identified as the receptor for systemin41.
Systemin plays a critical role in defense signaling in tomato. It promotes the synthesis of over 20 defense related proteins, mainly antinutritional proteins, signaling pathway proteins and proteases36. The over-expression of the prosystemin resulted in a significant decrease of the larvae damage, indicating that a high level of constitutive protection is superior to an inducible defense mechanism42. However, the continuous activation of prosystemin is costly, affecting the growth, the physiology and the reproductive success of tomato plants43. When systemin was silenced, production of protease inhibitors in tomato was severely impaired and larvae feeding on the plants grew three time faster44. Over expression of systemin has been found to improve the tolerance of plants to abiotic stress, including salt stress and UV radiation45. Systemin transgenic plants had higher stomatal conductances, lower leaf concentrations of abscissic acid and proline and a higher biomass when grown in salt solution. These findings suggest that systemin either allowed the plants to adapt to salt stress more efficiently or that they perceived a less stressful environment45.
When plants parallel grown under UVB light and normal condition (without UVB radiation) it is found that plants exposing UVB light are more resistant to insect herbivory comparatively plants grown under normal condition. When tomato plants are exposed to a pulse of UVB radiation and then weakly wounded protease inhibitors accumulate throughout the plant. By themselves, neither the radiation nor weak wounding is sufficient to induce systemic protease inhibitors accumulation. In Tomato cell cultures MAPKs are activated by both systemin and UVB acting together. Short pulse of UVB also causes alkalization of the culturing medium37,45. Beside these, systemin also increases root growth in Solanum pimpinellifolium suggesting that it may also play some role in plant development46.
As in normal cases the low cell density inhibited proliferation of fully differentiated mesophyll cells, growth of those cells was significantly promoted by adding conditioned medium derived from Asparagus cell culture32. Using this bioassay system, the active factor was purified from the conditioned medium and identified as a sulfated peptide composed of only five amino acids. Due to sulfated easters, the peptide was named phytosulfokine32. Phytosulfokine is produced by enzymatic processing of a ~80 amino acid precursor that has a secretion signal at its N terminal46. Phytosulfokine precursor genes are redundantly distributed throughout the genome, and are found in condition medium derived from cell line of many angiosperms and gymnosperms plant species including Asparagus, rice and maize, Zinnia, carrot and Arabidopsis, indicating that it is widely distributed among higher plants31,48-52.
Phytosulfokine containing two post translationally sulfated tyrosine residue and results from cleavage of the precursor proteins. Tyrosine sulfation of the preprophytosulfokine is catalyzed by the tyrosilprotein sulfotransferase in the golgi apparatus52-53. Research findings indicate that the LRR-RLK is a component of a functional phytosulfokine receptor that directly interacts with phytosulfokine54. The phytosulfokine binding LRR-RLK is named PSKR1. Expression of PSKR1 has been detected throughout the tissue of the leaves, apical meristem, hypocotyls, and root of carrot seedlings although much higher expression has been detected in cultured carrot cells31.
During a search for systemin in tobacco, an unrelated 49 amino acid peptide factor was discovered that alkalinizes suspension culture medium even more rapidly than systemins but does not activate defense response55-56. So this factor was named as RALF, for rapid alkalinazation factor. Beside tobacco, highly conserved similar sequence has been also isolated from the tomato and alfalfa leaves56. A synthetic peptide based on the tomato sequence was produced and it is found that both native and oxidized synthetic peptides were able to produce alkalinization responses at nanomolar concentrations, but alkylation of the reduced form of the peptide (which prevents formation of disulfide bridges) inactivated the synthetic peptide57.
Role of RALFs in defense was considered since both systemin and RALFs induce alkalinization of medium of MAP kinase activation55. Overexpression of RALF genes can inhibit overall plant growth or specific aspects of plant growth, depending on the targeted RALF gene. Transgenic studies in Arabidopsis thaliana showed that overexpression of either AtRALF1 or AtRALF23 resulted in semi-dwarf plants58-59. In Madicago trunculata, a root-expressed RALF gene (MtRALF1) was discovered as being upregulated by nodulation factors60. Overexpression of the MtRALF1 gene in transgenic plants resulted in a reduction in nodules and an increase in aborted infection threads. So, all of these studies support a role for RALF peptides in negatively regulating cell growth, in particular by inhibiting cell expansion57.
The aboveground body of higher plants is a consequence of the continual activity of the shoot apical meristem (SAM). Based on their function SAM can be divided into three zones: peripheral zone (PZ) which form the lateral organs; rib zone (RZ), forms the stem core; central zone (CZ), characterized by slow cell division and is the source of cells for the PZ and RZ. In the SAM of plants, a balance between the divisions of stem cells (central zone) and differentiating cells (peripheral zone) must be maintained. CLV genes are fundamental in maintaining the balance of cells in each zone8. The CLV genes derived their name from the latin word clavata (for club shape) because floral meristems of these mutants from numerous extra club like carpels1. Loss of function mutations in the Arabidopsis CLV1, CLV2 and CLV3 genes dramatically modify inflorescence architecture by enlarging the size of their SAMs61-62.
The CLV3 gene was identified as a specific regulator of shoot and floral meristems in 1995. In 1999, Fletcher et al. identified CLV3 as a small signaling peptide. CLV3 is specifically expressed in the L1-L3 stem cell layers of the central zone, while CLV1 is only expressed in deeper layers of the L3. This led to the conclusion that the CLV3 peptide could be secreted from the stem cells where it is produced and binds to the CLV1 or CLV2/CRN receptor complexes in L3 cells12. CLV3 has been suggested as an extracellular signaling polypeptide that is responsible for the determination of cell fate in the SAM, but its chemical structure is unknown63-64.
CLV proteins normally function to restrict WUS expression from the stem cell domain. Accordingly, WUS overexpression causes meristem overgrowth, resulting in plants that resemble clv mutants65. Some of the chemically synthesized CLV3 peptides, CLV3L, CLV3-A, CLV3-B, MCLV3, and MCLV3′, reduce the SAM and root apical meristem (RAM) size, which was consistent with the fact that overexpression of the CLV3 gene reduces SAM and RAM size8. The reduce SAM and RAM size indicate that activation of CLV like signaling pathway may also control cell fate in roots and that the CLV like peptide might also be involve in regulating RAM growth1,
In most leguminous plants nitrogen fixing bacteria (Rhizobia) take part in root nodule formation. Rhizobia induce nodule development on the plant by producing lipo-chito oligosaccharides known as Nod factors. Noduline genes are classified into two classes first as early nodulin (ENOD) and second as late nodulin (LNOD). The ENOD40 was the second polypeptide identified in plants and the first to be deduced using gene analysis56. It is proposed that ENOD40 encodes signaling peptides of 9-24 amino acids67. ENOD40 is induced long before the onset of cell division of root cortical cells by Nod factors from rhizobium bacteria, suggesting that ENOD40 is involved in nodule formation in legumes. However, homologs of ENOD40 have been isolated from not only legumes, but also nonlegumes including monocots, implying that ENOD40 may also have a function in plant developmental steps other than nodule organogenesis68-72.
Knockdown of ENOD40s leads to significant suppression of nodule formation in Lotus japonica and its overexpression accelerates nodulation in Medicago73. Such studied suggesting that this gene plays a main role in nodule development. However overexpression of ENOD40 has no effect on apparent aberration plant growth, this indicating that this gene does not directly trigger cell division, but rather sensitizes cells to division inducing signals1. Expression of ENOD has also been observed in nonsymbiotic organs of legumes and homologs have been found in nonlegumes68,74-75. In rice, ENOD40 transcription is restricted to parenchyma cells surrounding the protoxylem, during early development of lateral vascular bundles that conjoin an emerging leaf. This indicates that ENOD40 peptides might play an important role in vascular bundle development67,76. All these research findings suggesting that ENOD40 was originally involved in another plant developmental pathway, and was then employ into the symbiotic nodulation pathway.
In many flowering plants pollen from the closely related individuals is recognized and rejected by the pistil to prevent inbreeding and maintain genetic diversity within a species, this is known as self-incompatibility. It is revealed through genetic studies that self incompatibility is controlled by a single multiallelic locus named as sterility locus (S-locus)77. A highly polymorphic, small and anther-specific gene was discovered which controls pollen function in self incompatibility. This gene was located between S-locus receptor like-kinase (SRK) and S-locus glycoprotein (SLG) at the S-locus and known as S locus cysteine-rich protein (SCR) or S locus protein 11 (SP11) 23,78. Self-incompatibility determinants in Brassica species have been identified through molecular cloning of S-locus genes. The products of S locus genes are expressed specifically in the stigma, pollen or anther1.
Research finding suggest that the SCR/SP11 gene product is necessary and sufficient to determine pollen self incompatibility. Immuno-histochemical experiments suggest that at the early developmental stage of the anther, SP11 is secreted from the tapetal cell into the anther locule as a cluster and translocated to the pollen surface. During the pollination process, SP11 is translocated from the pollen surface to the papilla cell and then penetrates the cuticle layer of the papilla cell to diffuse across the pectin cellulose layer79-80. The pollen of Brassica plants transformed with a certain SCR/SP11 haplotype acquired the self-incompatibility specificity encoded by the transgene. Recombinant or chemically synthesized SCR/SP11 peptide applied to the stigma at concentrations as low as 50 fmol per stigma inhibited the hydration of compatible pollen78.
The POLARIS gene was identified in an exhaustive analysis of a promoter trap transgenic line in which reporter gene expression is observed predominantly in roots. Expression of PLS has been detected in embryonic root from the heart stage and in seedling primary and lateral root tips81. Open reading frame is located within a short (~500 nucleotides) auxin inducible transcript and encodes a predicted polypeptide of 36 amino acid residues82. The suggested 36 amino acid peptide has no secretion signal; this indicated that its functions are in the cytoplasm. However there is no direct evidence to prove its intracellular localization. There are many research efforts made to isolate PLS peptide but it still has not biochemically isolated1.
The pls mutants have a short-root phenotype and reduced vascularization of leaves due to reduced cell expansion and increased radial expansion; pls mutants roots are hyper responsive to exogenous cytokinins and show increased expression of the cytokinin-inducible gene ARR5/IBC6 compared with the wild type76. Overexpression of PLS reduces inhibition of root growth by exogenous cytokinins and increases leaf vascularization1.
Abscission is a physiological process of cell separation that enables plants to shed unwanted organs such as old leaves or floral organs from the parent plant body. Abscission begins with formation of the abscission zone, which divides the plant body from the organs to be shed. During screening for mutants with delayed floral abscission an Arabidopsis mutant called ida was identified which retains it floral organs indefinitely83. In ida mutant plants the senesced dry floral parts live attached, even after the shedding of mature seeds. A 77 amino acid peptide with an N-terminal secretion signal peptide is encoded by the IDA gene. Promoter study has revealed that during the floral abscission process, IDA is expressed in the floral organ abscission zone. In Arabidopsis, the research findings have also identified 5 genes paralogous to IDA, IDl1-5. Sequence alignments of the deduce peptides of this family show the presence of a highly conserved domain flanked by basic amino acid residues near the C terminal1.
The available evidence suggests that IDA encodes a small peptide which is a ligand for HAESA. This small peptide is an Arabidopsis plasma membrane-associated LRR-RLK which is involved in controlling floral organ abscission84. HAESA is expressed not only at the base of petioles and pedicels but also in abscission zones of floral organs. However the IDA peptides are mostly known for their role in floral organ abscission83. But recently a new function has been assign to facilitate the passage of lateral root primordial through the main root and to assist in the lateral root85.
During the screening of activation tagged populations of Arabidopsis for isolation of leaf shape mutants, a new gene was identified which is known as ROTUNDIFOLIA (ROT4)86. While at around the same time another gene known as DEVIL1 (DVL1) was identified in an activation pool87. rot4-1D is a dominant mutant that express misexpression of ROT4 due to insertion of T-DNA carrying 35S enhancers, and rot4-1D mutant plants have short and rounded leaves, short floral organs, and short inflorescence stems. The phenotype is due to reduced cell proliferation, specifically in the longitudinal axis of the organs, suggesting that the ROT4 gene controls polar cell proliferation. While dvl1-1D has rounder leaves, shortened petioles, shortened siliques, and moderately horned fruit tips. However, this phenotype is very similar to that of rot4-1D1.
The ROT4 and DVL1 genes have small ORFs that encode a 53 and 51 amino acid peptides respectively. The amino acid sequences of both are highly homologous to each other. Database searches indicate that ROT4 and DVL1 are members of an Arabidopsis gene family containing 23 small genes that potentially encode short peptides. Overexpression of the ROT4-GFP fusion rescues the rot4-1D phenotype, suggesting that expression of this small ORF is sufficient for its function. Functional redundancy of the ROT4/DVL1 family is also consistent with the finding that disruption of only one member of the family causes no obvious phenotypic change. Like ROT4, DVL1 is expressed in leaves, whereas some other members of the ROT4/DVL1 are family expressed in flowers and roots1.
Six lipophilic nonpeptide plant hormones are well studied in plants than peptide hormones because of their important role in plant development and growth. However, now it is clear that peptide hormones are also play very crucial role in intercellular and intracellular signaling in plants. More than 1,000 genes have been found in the complete genome sequence of Arabidopsis that encode putative secreted peptides with a potential signaling function17,88. Combining the plant genome database with in silico bioinformatics analysis can be effective in identifying new signaling peptide genes potentially involved in signal transduction.
There are many aspect remain unexposed about the peptide hormones in plants. The information on the biosynthesis of the peptide hormones is very little, only a few proteases have been reported to be involved in the processing of the precursors so it is very hard to explain how they are produced in plants. Their rout of from the initial translation to the secretion of mature peptide is still hypothetical59,89-90. The role of plant peptide hormone in controlling plant physiology, growth and development through various processes are studied but in most cases, the receptors they bind to and the downstream targets are unknown. It is expected that ongoing researches on forward and reverse genetic studies will surely provide some imminent knowledge regarding this in the near future.