Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or ...damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment.
ENOD40 is expressed at an early stage in root nodule organogenesis in legumes. Identification of ENOD40 homologs in nonleguminous plants suggests that this gene may have a more general biological ...function. In vitro translation of soybean ENOD40 mRNA in wheat germ extracts revealed that the conserved nucleotide sequence at the 5′ end (region I) encodes two peptides of 12 and 24 aa residues (peptides A and B). These peptides are synthesized de novo from very short, overlapping ORFs. Appropriate ORFs are present in all legume ENOD40s studied thus far. In this case small peptides are directly translated from polycistronic eukaryotic mRNA. The 24-aa peptide B was detected in nodules by Western blotting. Both peptides specifically bind to the same 93-kDa protein, which was affinity purified from soybean nodules. Using peptide mass fingerprinting, we identified this binding protein as nodulin 100, which is a subunit of sucrose synthase. Based on our data we suggest that ENOD40 peptides are involved in the control of sucrose use in nitrogen-fixing nodules.
The late embryogenesis abundant (LEA)-like protein CDeT11-24 is one of the major desiccation-related phosphoproteins of the resurrection plant Craterostigma plantagineum. In this study, it was shown ...that CDeT11-24 is mostly intrinsically disordered and protects two different enzymes, citrate synthase and lactate dehydrogenase, against damaging effects caused by desiccation. Lipid-binding assays revealed that CDeT11-24 is able to interact with phosphatidic acid, although electrostatic repulsion was expected due to the overall negative net charge of the protein under the tested physiological conditions. CDeT11-24 carries an N-terminal lysine-rich sequence, which is predicted to form an amphipathic α-helix. Analysis of the truncated CDeT11-24 protein identified this region to be responsible for both activities: enzyme protection and phosphatidic acid interaction. Possible functions of the CDeT11-24 protein are discussed in the context of desiccation tolerance.
ABSTRACT
Reversible phosphorylation of proteins is an important mechanism by which organisms regulate their reactions to external stimuli. To investigate the involvement of phosphorylation during ...acquisition of desiccation tolerance, we have analysed dehydration‐induced protein phosphorylation in the desiccation tolerant resurrection plant Craterostigma plantagineum. Several dehydration‐induced proteins were shown to be transiently phosphorylated during a dehydration and rehydration (RH) cycle. Two abundantly expressed phosphoproteins are the dehydration‐ and abscisic acid (ABA)‐responsive protein CDeT11‐24 and the group 2 late embryogenesis abundant (LEA) protein CDeT6‐19. Although both proteins accumulate in leaves and roots with similar kinetics in response to dehydration, their phosphorylation patterns differ. Several phosphorylation sites were identified on the CDeT11‐24 protein using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The coincidence of phosphorylation sites with predicted coiled‐coil regions leads to the hypothesis that CDeT11‐24 phosphorylations influence the stability of coiled‐coil interactions with itself and possibly other proteins.
Summary
Phospholipase D (PLD) and its cleavage product phosphatidic acid (PA) are crucial in plant stress‐signalling. Although some targets of PLD and PA have been identified, the signalling pathway ...is still enigmatic. This study demonstrates that the phosphoprotein At5g39570, now called PLD‐regulated protein1 (PLDrp1), from Arabidopsis thaliana is directly regulated by PLDα1. The protein PLDrp1 can be divided into two regions with distinct properties. The conserved N‐terminal region specifically binds PA, while the repeat‐rich C‐terminal domain suggests interactions with RNAs. The expression of PLDrp1 depends on PLDα1 and the plant water status. Water stress triggers a pldα1‐like phenotype in PLDrp1 mutants and induces the expression of PLDrp1 in pldα1 mutants. The regulation of PLDrp1 by PLDα1 and environmental stressors contributes to the understanding of the complex PLD regulatory network and presents a new member of the PA‐signalling chain in plants.
Significance Statement
This manuscript reports PLDrp1 protein as a target of PLD. This protein specifically binds to phosphatidic acid.
Summary
Phospholipase D (
PLD
) and its cleavage product phosphatidic acid (
PA
) are crucial in plant stress‐signalling. Although some targets of
PLD
and
PA
have been identified, the signalling ...pathway is still enigmatic. This study demonstrates that the phosphoprotein At5g39570, now called
PLD
‐regulated protein1 (
PLD
rp1), from
Arabidopsis thaliana
is directly regulated by
PLD
α1. The protein
PLD
rp1 can be divided into two regions with distinct properties. The conserved N‐terminal region specifically binds
PA
, while the repeat‐rich C‐terminal domain suggests interactions with
RNA
s. The expression of
PLD
rp1 depends on
PLD
α1 and the plant water status. Water stress triggers a
pld
α
1
‐like phenotype in
PLD
rp1
mutants and induces the expression of
PLD
rp1 in
pld
α
1
mutants. The regulation of
PLD
rp1 by
PLD
α1 and environmental stressors contributes to the understanding of the complex
PLD
regulatory network and presents a new member of the
PA
‐signalling chain in plants.
Significance Statement
This manuscript reports PLDrp1 protein as a target of PLD. This protein specifically binds to phosphatidic acid.
The common nodulation genes nodABC are conserved in all rhizobia and are involved in synthesis of a lipooligosaccharide signal molecule. This bacterial signal consists of a chitooligosaccharide ...backbone, which carries at the nonreducing end a fatty acyl chain. The modified chitooligosac-charide molecule triggers development of nodules on the roots of the leguminous host plant. To elucidate the specific role of the NodB protein in nodulation factor synthesis, we have purified recombinant NodB and determined its biochemical role by direct assays. Our data show that the NodB protein of Rhizobium meliloti deacetylates the nonreducing N-acetylglu-cosamine residue of chitooligosaccharides. The monosaccharide N-acetylglucosamine is not deacetylated by NodB. In the pathway of Nod factor synthesis, deacetylation at the nonreducing end of the oligosaccharide backbone may be a necessary requirement for attachment of the fatty acyl chain.
Rhizobium meliloti interacts symbiotically with alfalfa by forming root nodules in which the bacteria fix nitrogen. The Rhizobium nodulation genes nodABC are involved in the synthesis of ...lipooligosaccharide symbiotic signal molecules, which are mono-N-acylated chitooligosaccharides. These bacterial signals elicit nodule organogenesis in roots of legumes. To elucidate the role of the NodA protein in lipooligosaccharide biosynthesis, we prepared a radiolabeled tetrasaccharide precursor carrying an amino group as a potential attachment site for N-acylation at the nonreducing glucosamine residue. Various criteria demonstrate that NodA is involved in the attachment of a fatty acyl chain to this tetrasaccharide precursor, yielding a biologically active nodulation factor.
Reversible protein phosphorylation/dephosphorylation is crucial for regulation of many cellular events, and increasing evidence indicates that this post-translational modification is also involved in ...the complex process of acquisition of desiccation tolerance. To analyze the phosphoproteome of the desiccation tolerant resurrection plant Craterostigma plantagineum, MOAC-enriched proteins from leaves at different stages of a de-/rehydration cycle were separated by 2-D PAGE and detected by phosphoprotein-specific staining. Using this strategy 20 putative phosphoproteins were identified by MALDI-TOF MS and MS/MS, which were not detected when total proteins were analyzed. The characterized desiccation-related phosphoproteins CDeT11-24 and CDeT6-19 were used as internal markers to validate the specificity of the analyses. For 16 of the identified proteins published evidence suggests that they are phosphoproteins. Comparative analysis of the 2-D gels showed that spot intensities of most identified putative phosphoproteins change during the de-/rehydration cycle. This suggests an involvement of these proteins in desiccation tolerance. Nearly all changes in the phosphoproteome of C. plantagineum, which are triggered by dehydration, are reversed within 4 days of rehydration, which is in agreement with physiological observations. Possible functions of selected proteins are discussed in the context of the de-/rehydration cycle.
Nodulation (Nod) factors are lipo-chitooligosaccharides (LCOs) secreted by rhizobia to trigger the early steps of nodule organogenesis in leguminous plants. A method to synthesize LCOs in vitro was ...developed. Synthetic LCOs alleviated the requirement for auxin and cytokinin to sustain growth of cultured tobacco protoplasts. LCOs containing C$_{18:1}$ trans-fatty acyl substituents were more effective than those containing cis-fatty acids in promoting cell division as well as in activating an auxin-responsive promoter and the expression of a gene implicated in auxin action. These data indicate that LCOs redirect plant growth also in nonlegumes by activating developmental pathways also targeted by phytohormones.
PDF
