Internalization of miPEP165a into Arabidopsis Roots Depends on Both Passive Diffusion and Endocytosis-Associated Processes.

MiPEPs are short natural peptides encoded by microRNAs in plants. Exogenous application of miPEPs increases the expression of their corresponding miRNA and, consequently, induces consistent phenotypical changes. Therefore, miPEPs carry huge potential in agronomy as gene regulators that do not require genome manipulation. However, to this end, it is necessary to know their mode of action, including where they act and how they enter the plants. Here, after analyzing the effect of Arabidopsis thaliana miPEP165a on root and aerial part development, we followed the internalization of fluorescent-labelled miPEP165a into roots and compared its uptake into endocytosis-altered mutants to that observed in wild-type plants treated or not with endocytosis inhibitors. The results show that entry of miPEP165a involves both a passive diffusion at the root apex and endocytosis-associated internalization in the differentiation and mature zones. Moreover, miPEP165a is unable to enter the central cylinder and does not migrate from the roots to the aerial part of the plant, suggesting that miPEPs have no systemic effect.

Calcium- and Nitric Oxide-Dependent Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Response to Long Chain Bases in Tobacco BY-2 Cells.

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid long chain bases (LCBs) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in plants. In addition, in tobacco BY-2 cells, it has been shown that DHS triggers a rapid production of H2O2 and nitric oxide (NO). Recently, in analogy to what is known in the animal field, plant cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a ubiquitous enzyme involved in glycolysis, has been suggested to fulfill other functions associated with its oxidative post-translational modifications such as S-nitrosylation on cysteine residues. In particular, in mammals, stress signals inducing NO production promote S-nitrosylation of GAPC and its subsequent translocation into the nucleus where the protein participates in the establishment of apoptosis. In the present study, we investigated the behavior of GAPC in tobacco BY-2 cells treated with DHS. We found that upon DHS treatment, an S-nitrosylated form of GAPC accumulated in the nucleus. This accumulation was dependent on NO production. Two genes encoding GAPCs, namely Nt(BY-2)GAPC1 and Nt(BY-2)GAPC2, were cloned. Transient overexpression of Nt(BY-2)GAPC-green fluorescent protein (GFP) chimeric constructs indicated that both proteins localized in the cytoplasm as well as in the nucleus. Mutating into serine the two cysteine residues thought to be S-nitrosylated in response to DHS did not modify the localization of the proteins, suggesting that S-nitrosylation of GAPCs was probably not necessary for their nuclear relocalization. Interestingly, using Förster resonance energy transfer experiments, we showed that Nt(BY-2)GAPCs interact with nucleic acids in the nucleus. When GAPCs were mutated on their cysteine residues, their interaction with nucleic acids was abolished, suggesting a role for GAPCs in the protection of nucleic acids against oxidative stress.

The Essentials on microRNA-Encoded Peptides from Plants to Animals.

Primary transcripts of microRNAs (pri-miRNAs) were initially defined as long non-coding RNAs that host miRNAs further processed by the microRNA processor complex. A few years ago, however, it was discovered in plants that pri-miRNAs actually contain functional open reading frames (sORFs) that translate into small peptides called miPEPs, for microRNA-encoded peptides. Initially detected in Arabidopsis thaliana and Medicago truncatula, recent studies have revealed the presence of miPEPs in other pri-miRNAs as well as in other species ranging from various plant species to animals. This suggests that miPEP numbers remain largely underestimated and that they could be a common signature of pri-miRNAs. Here we present the most recent advances in miPEPs research and discuss how their discovery has broadened our vision of the regulation of gene expression by miRNAs, and how miPEPs could be interesting tools in sustainable agriculture or the treatment of certain human diseases.

Complementary peptides represent a credible alternative to agrochemicals by activating translation of targeted proteins.

The current agriculture main challenge is to maintain food production while facing multiple threats such as increasing world population, temperature increase, lack of agrochemicals due to health issues and uprising of weeds resistant to herbicides. Developing novel, alternative, and safe methods is hence of paramount importance. Here, we show that complementary peptides (cPEPs) from any gene can be designed to target specifically plant coding genes. External application of synthetic peptides increases the abundance of the targeted protein, leading to related phenotypes. Moreover, we provide evidence that cPEPs can be powerful tools in agronomy to improve plant traits, such as growth, resistance to pathogen or heat stress, without the needs of genetic approaches. Finally, by combining their activity they can also be used to reduce weed growth.

Characterization of plant microRNA-encoded peptides (miPEPs) reveals molecular mechanisms from the translation to activity and specificity.

MicroRNAs (miRNAs) are transcribed as long primary transcripts (pri-miRNAs) by RNA polymerase II. Plant pri-miRNAs encode regulatory peptides called miPEPs, which specifically enhance the transcription of the pri-miRNA from which they originate. However, paradoxically, whereas miPEPs have been identified in different plant species, they are poorly conserved, raising the question of the mechanisms underlying their specificity. To address this point, we identify and re-annotate multiple Arabidopsis thaliana pri-miRNAs in order to identify ORF encoding miPEPs. The study of several identified miPEPs in different species show that non-conserved miPEPs are only active in their plant of origin, whereas conserved ones are active in different species. Finally, we find that miPEP activity relies on the presence of its own miORF, explaining both the lack of selection pressure on miPEP sequence and the ability for non-conserved peptides to play a similar role, i.e., to activate the expression of their corresponding miRNA.

Sphingolipid-induced cell death in Arabidopsis is negatively regulated by the papain-like cysteine protease RD21.

It is now well established that sphingoid Long Chain Bases (LCBs) are crucial mediators of programmed cell death. In plants, the mycotoxin fumonisin B1 (FB1) produced by the necrotrophic fungus Fusarium moniliforme disrupts the sphingolipid biosynthesis pathway by inhibiting the ceramide synthase leading to an increase in the amount of phytosphingosine (PHS) and dihydrosphingosine (DHS), the two major LCBs in Arabidopsis thaliana. To date, the signaling pathway involved in FB1-induced cell death remains largely uncharacterized. It is also well acknowledged that plant proteases such as papain-like cysteine protease are largely involved in plant immunity. Here, we show that the papain-like cysteine protease RD21 (responsive-to-desiccation-21) is activated in response to PHS and FB1 in Arabidopsis cultured cells and leaves, respectively. Using two allelic null mutants of RD21, and two different PCD bioassays, we demonstrate that the protein acts as a negative regulator of FB1-induced cell death in Arabidopsis.

CDPKs and 14-3-3 Proteins: Emerging Duo in Signaling.

Calcium-dependent protein kinases (CDPKs) are Ca2+-sensors that play pivotal roles in plant development and stress responses. They have the unique ability to directly translate intracellular Ca2+ signals into reversible phosphorylation events of diverse substrates which can mediate interactions with 14-3-3 proteins to modulate protein functions. Recent studies have revealed roles for the coordinated action of CDPKs and 14-3-3s in regulating diverse aspects of plant biology including metabolism, development, and stress responses. We review here the underlying interaction and cross-regulation of the two signaling proteins, and we discuss how this insight has led to the emerging concept of CDPK/14-3-3 signaling modules that could contribute to response specificity.

Regulation of the wheat MAP kinase phosphatase 1 by 14-3-3 proteins.

Plant MAP kinase phosphatases (MKPs) are major regulators of MAPK signaling pathways and play crucial roles in controlling growth, development and stress responses. The presence of several functional domains in plant MKPs such as a dual specificity phosphatase catalytic domain, gelsolin, calmodulin-binding and serine-rich domains, suggests that MKPs can interact with distinct cellular partners, others than MAPKs. In this report, we identified a canonical mode I 14-3-3-binding motif (574KLPSLP579) located at the carboxy-terminal region of the wheat MKP, TMKP1. We found that this motif is well-conserved among other MKPs from monocots including Hordeum vulgare, Brachypodium distachyon and Aegilops taushii. Using co-immunoprecipitation assays, we provide evidence for interaction between TMKP1 and 14-3-3 proteins in wheat. Moreover, the phosphatase activity of TMKP1 is increased in a phospho-dependent manner by either Arabidopsis or yeast 14-3-3 isoforms. TMKP1 activation by 14-3-3 proteins is enhanced by Mn2+, whereas in the presence of Ca2+ ions, TMKP1 activation was limited to Arabidopsis 14-3-3φ (phi), an isoform harboring an EF-hand motif. Such findings strongly suggest that 14-3-3 proteins, in conjunction with specific divalent cations, may stimulate TMKP1 activity and point-out that 14-3-3 proteins bind and regulate the activity of a MKP in eukaryotes.