Identification and Characterization of VDAC Family in Maize.

The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane (OMM) of all eukaryotes, having an important role in the communication between mitochondria and cytosol. The plant VDAC family consists of a wide variety of members that may participate in cell responses to several environmental stresses. However, there is no experimental information about the members comprising the maize VDAC (ZmVDAC) family. In this study, the ZmVDAC family was identified, and described, and its gene transcription profile was explored during the first six days of germination and under different biotic stress stimuli. Nine members were proposed as bona fide VDAC genes with a high potential to code functional VDAC proteins. Each member of the ZmVDAC family was characterized in silico, and nomenclature was proposed according to phylogenetic relationships. Transcript levels in coleoptiles showed a different pattern of expression for each ZmVDAC gene, suggesting specific roles for each one during seedling development. This expression profile changed under Fusarium verticillioides infection and salicylic acid, methyl jasmonate, and gibberellic acid treatments, suggesting no redundancy for the nine ZmVDAC genes and, thus, probably specific and diverse functions according to plant needs and environmental conditions. Nevertheless, ZmVDAC4b was significantly upregulated upon biotic stress signals, suggesting this gene's potential role during the biotic stress response.

MITOGEN-ACTIVATED PROTEIN KINASE PHOSPHATASE 1 mediates root sensing of serotonin through jasmonic acid signaling and modulating reactive oxygen species.

Serotonin (5-hydroxytryptamine) acts as a neurotransmitter in mammals and is widely distributed in the plant kingdom, where it influences root growth and defense. Mitogen-Activated Protein Kinases (MAPKs) and MAPK phosphatases (MKPs) play critical functions in decoding hormonal signalling, but their possible roles in mediating serotonin responses await investigation. In this report, we unveiled positive roles for the MITOGEN-ACTIVATED PROTEIN KINASE PHOSPHATASE1 (MKP1) in the inhibition of the primary root growth, cell division, meristem structure, and differentiation events in Arabidopsis seedlings. mkp1 mutants were less sensitive to jasmonic acid applications that halted primary root growth in wild-type (WT) plants, and consistently, the neurotransmitter activated the expression of the JASMONATE ZIM-domain (JAZ) proteins JAZ1 and JAZ10, two critical proteins orchestrating jasmonic acid signalling. This effect correlated with exacerbated production of endogenous reactive oxygen species (ROS) in the WT, a process constitutively manifested in mkp1 mutants. These data help to clarify the relationship between serotonin and growth/defense trade-offs, and reveal the importance of the MAPK pathway in root development through ROS production.

Bacterial cyclodipeptides elicit Arabidopsis thaliana immune responses reducing the pathogenic effects of Pseudomonas aeruginosa PAO1 strains on plant development.

Plants being sessile organisms are exposed to various biotic and abiotic factors, thus causing stress. The Pseudomonas aeruginosa bacterium is an opportunistic pathogen for animals, insects, and plants. Direct exposure of Arabidopsis thaliana to the P. aeruginosa PAO1 strain induces plant death by producing a wide variety of virulence factors, which are regulated mainly by quorum sensing systems. Besides virulence factors, P. aeruginosa PAO1 also produces cyclodipeptides (CDPs), which possess auxin-like activity and promote plant growth through activation of the target of the rapamycin (AtTOR) pathway. On the other hand, plant defense mechanisms are regulated through the production of phytohormones, such as salicylic acid (SA) and jasmonic acid (JA), which are induced in response to pathogen-associated molecular patterns (PAMPs), activating defense genes associated with SA and JA such as PATHOGENESIS-RELATED-1 (PR-1) and LIPOXYGENASE2 (LOX2), respectively. PR proteins are suggested to play critical roles in coordinating the Systemic Acquired Resistance (SAR). In contrast, LOX proteins (LOX2, LOX3, and LOX4) have been associated with the production of JA by producing its precursors, oxylipins. The activation of defense mechanisms involves signaling cascades such as Mitogen-Activated Protein Kinases (MAPKs) or the TOR pathway as a switch for re-directing energy towards defense or growth. In this work, we challenged A. thaliana (wild type, mpk6 or mpk3 mutants, and overexpressing TOR) seedlings with P. aeruginosa PAO1 strains to identify the role of bacterial CDPs in the plant immune response. Results showed that the pre-exposure of these Arabidopsis seedlings to CDPs significantly reduced plant infection of the pathogenic P. aeruginosa PAO1 strains, indicating that plants that over-express AtTOR or lack MPK3/MPK6 protein-kinases are more susceptible to the pathogenic effects. In addition, CDPs induced the GUS activity only in the LOX2::GUS plants, indicative of JA-signaling activation. Our findings indicate that the CDPs are molecules that trigger SA-independent and JA-dependent defense responses in A. thaliana; hence, bacterial CDPs may be considered elicitors of the Arabidopsis immune response to pathogens.

MPK6 Kinase Regulates Plasma Membrane H+-ATPase Activity in Cold Acclimation.

Cold and freezing stresses severely affect plant growth, development, and survival rate. Some plant species have evolved a process known as cold acclimation, in which plants exposed to temperatures above 0 °C trigger biochemical and physiological changes to survive freezing. During this response, several signaling events are mediated by transducers, such as mitogen activated protein kinase (MAPK) cascades. Plasma membrane H+-ATPase is a key enzyme for the plant cell life under regular and stress conditions. Using wild type and mpk3 and mpk6 knock out mutants in Arabidopsis thaliana, we explored the transcriptional, translational, and 14-3-3 protein regulation of the plasma membrane H+-ATPase activity under the acclimation process. The kinetic analysis revealed a differential profiling of the H+-ATPase activity depending on the presence or absence of MPK3 or MPK6 under non-acclimated or acclimated conditions. Negative regulation of the plasma membrane H+-ATPase activity was found to be exerted by MPK3 in non-acclimated conditions and by MPK6 in acclimated conditions, describing a novel form of regulation of this master ATPase. The MPK6 regulation involved changes in plasma membrane fluidity. Moreover, our results indicated that MPK6 is a critical regulator in the process of cold acclimation that leads to freezing tolerance and further survival.

A phylogenetic study of the members of the MAPK and MEK families across Viridiplantae.

Protein phosphorylation is regulated by the activity of enzymes generically known as kinases. One of those kinases is Mitogen-Activated Protein Kinases (MAPK), which operate through a phosphorylation cascade conformed by members from three related protein kinase families namely MAPK kinase kinase (MEKK), MAPK kinase (MEK), and MAPK; these three acts hierarchically. Establishing the evolution of these proteins in the plant kingdom is an interesting but complicated task because the current MAPK, MAPKK, and MAPKKK subfamilies arose from duplications and subsequent sub-functionalization during the early stage of the emergence of Viridiplantae. Here, an in silico genomic analysis was performed on 18 different plant species, which resulted in the identification of 96 genes not previously annotated as components of the MAPK (70) and MEK (26) families. Interestingly, a deeper analysis of the sequences encoded by such genes revealed the existence of putative domains not previously described as signatures of MAPK and MEK kinases. Additionally, our analysis also suggests the presence of conserved activation motifs besides the canonical TEY and TDY domains, which characterize the MAPK family.

The growth of Arabidopsis primary root is repressed by several and diverse amino acids through auxin-dependent and independent mechanisms and MPK6 kinase activity.

Amino acids serve as structural monomers for protein synthesis and are considered important biostimulants for plants. In this report, the effects of all 20-L amino acids in Arabidopsis primary root growth were evaluated. 15 amino acids inhibited growth, being l-leucine (l-Leu), l-lysine (l-Lys), l-tryptophan (l-Trp), and l-glutamate (l-Glu) the most active, which repressed both cell division and elongation in primary roots. Comparisons of DR5:GFP expression and growth of WT Arabidopsis seedlings and several auxin response mutants including slr, axr1 and axr2 single mutants, arf7/arf19 double mutant and tir1/afb2/afb3 triple mutant, treated with inhibitory concentrations of l-Glu, l-Leu, l-Lys and l-Trp revealed gene-dependent, specific changes in auxin response. In addition, l- isomers of Glu, Leu and Lys, but not l-Trp diminished the GFP fluorescence of pPIN1::PIN1:GFP, pPIN2::PIN2:GFP, pPIN3::PIN3:GFP and pPIN7::PIN7:GFP constructs in root tips. MPK6 activity in roots was enhanced by amino acid treatment, being greater in response to l-Trp while mpk6 mutants supported cell division and elongation at high doses of l-Glu, l-Leu, l-Lys and l-Trp. We conclude that independently of their auxin modulating properties, amino acids signals converge in MPK6 to alter the Arabidopsis primary root growth.

High levels of glucose alter Physcomitrella patens metabolism and trigger a differential proteomic response.

Sugars act not only as substrates for plant metabolism, but also have a pivotal role in signaling pathways. Glucose signaling has been widely studied in the vascular plant Arabidopsis thaliana, but it has remained unexplored in non-vascular species such as Physcomitrella patens. To investigate P. patens response to high glucose treatment, we explored the dynamic changes in metabolism and protein population by applying a metabolomic fingerprint analysis (DIESI-MS), carbohydrate and chlorophyll quantification, Fv/Fm determination and label-free untargeted proteomics. Glucose feeding causes specific changes in P. patens metabolomic fingerprint, carbohydrate contents and protein accumulation, which is clearly different from those of osmotically induced responses. The maximal rate of PSII was not affected although chlorophyll decreased in both treatments. The biological process, cellular component, and molecular function gene ontology (GO) classifications of the differentially expressed proteins indicate the translation process is the most represented category in response to glucose, followed by photosynthesis, cellular response to oxidative stress and protein refolding. Importantly, although several proteins have high fold changes, these proteins have no predicted identity. The most significant discovery of our study at the proteome level is that high glucose increase abundance of proteins related to the translation process, which was not previously evidenced in non-vascular plants, indicating that regulation by glucose at the translational level is a partially conserved response in both plant lineages. To our knowledge, this is the first time that metabolome fingerprint and proteomic analyses are performed after a high sugar treatment in non-vascular plants. These findings unravel evolutionarily shared and differential responses between vascular and non-vascular plants.

Bacillus velezensis 83 a bacterial strain from mango phyllosphere, useful for biological control and plant growth promotion.

Bacillus velezensis 83 was isolated from mango tree phyllosphere of orchards located in El Rosario, Sinaloa, México. The assessment of this strain as BCA (biological control agent), as well as PGPB (plant growth-promoting bacteria), were demonstrated through in vivo and in vitro assays. In vivo assays showed that B. velezensis 83 was able to control anthracnose (Kent mangoes) as efficiently as chemical treatment with Captan 50 PH™ or Cupravit hidro™. The inoculation of B. velezensis 83 to the roots of maize seedlings yielded an increase of 12% in height and 45% of root biomass, as compared with uninoculated seedlings. In vitro co-culture assays showed that B. velezensis 83 promoted Arabidopsis thaliana growth (root and shoot biomass) while, under the same experimental conditions, B. velezensis FZB42 (reference strain) had a suppressive effect on plant growth. In order to characterize the isolated strain, the complete genome sequence of B. velezensis 83 is reported. Its circular genome consists of 3,997,902 bp coding to 3949 predicted genes. The assembly and annotation of this genome revealed gene clusters related with plant-bacteria interaction and sporulation, as well as ten secondary metabolites biosynthetic gene clusters implicated in the biological control of phytopathogens. Despite the high genomic identity (> 98%) between B. velezensis 83 and B. velezensis FZB42, they are phenotypically different. Indeed, in vitro production of compounds such as surfactin and bacillomycin D (biocontrol activity) and γ-PGA (biofilm component) is significantly different between both strains.

YUCCA4 overexpression modulates auxin biosynthesis and transport and influences plant growth and development via crosstalk with abscisic acid in Arabidopsis thaliana.

Auxin regulates a plethora of events during plant growth and development, acting in concert with other phytohormones. YUCCA genes encode flavin monooxygenases that function in tryptophan-dependent auxin biosynthesis. To understand the contribution of the YUCCA4 (YUC4) gene on auxin homeostasis, plant growth and interaction with abscisic acid (ABA) signaling, 35S::YUC4 seedlings were generated, which showed elongated hypocotyls with hyponastic leaves and changes in root system architecture that correlate with enhanced auxin responsive gene expression. Differential expression of PIN1, 2, 3 and 7 auxin transporters was detected in roots of YUC4 overexpressing seedlings compared to the wild-type: PIN1 was down-regulated whereas PIN2, PIN3 and PIN7 were up-regulated. Noteworthy, 35S::YUC4 lines showed enhanced sensitivity to ABA on seed germination and post-embryonic root growth, involving ABI4 transcription factor. The auxin reporter genes DR5::GUS, DR5::GFP and BA3::GUS further revealed that abscisic acid impairs auxin responses in 35S::YUC4 seedlings. Our results indicate that YUC4 overexpression influences several aspects of auxin homeostasis and reveal the critical roles of ABI4 during auxin-ABA interaction in germination and primary root growth.

Mitogen-activated protein kinase 6 integrates phosphate and iron responses for indeterminate root growth in Arabidopsis thaliana.

MAIN CONCLUSION: A MAPK module, of which MPK6 kinase is an important component, is involved in the coordination of the responses to Pi and Fe in the primary root meristem of Arabidopsis thaliana. Phosphate (Pi) deficiency induces determinate primary root growth in Arabidopsis through cessation of cell division in the meristem, which is linked to an increased iron (Fe) accumulation. Here, we show that Mitogen-Activated Protein Kinase6 (MPK6) has a role in Arabidopsis primary root growth under low Pi stress. MPK6 activity is induced in roots in response to low Pi, and such induction is enhanced by Fe supplementation, suggesting an MPK6 role in coordinating Pi/Fe balance in mediating root growth. The differentiation of the root meristem induced by low Pi levels correlates with altered expression of auxin-inducible genes and auxin transporter levels via MPK6. Our results indicate a critical role of the MPK6 kinase in coordinating meristem cell activity to Pi and Fe availability for proper primary root growth.

Biochemical properties and subcellular localization of six members of the HXK family in maize and its metabolic contribution to embryo germination.

BACKGROUND: Seed germination is a crucial process in the plant life cycle when a dramatic variation of type and sugar content occurs just as the seed is hydrated. The production of hexose 6 phosphate is a key node in different pathways that are required for a successful germination. Hexokinase (HXK) is the only plant enzyme that phosphorylates glucose (Glc), so it is key to fueling several metabolic pathways depending on their substrate specificity, metabolite regulatory responses and subcellular localization. In maize, the HXK family is composed of nine genes, but only six of them (ZmHXK4-9) putatively encode catalytically active enzymes. Here, we cloned and functionally characterized putative catalytic enzymes to analyze their metabolic contribution during germination process. RESULTS: From the six HXKs analyzed here, only ZmHXK9 has minimal hexose phosphorylating activity even though enzymatic function of all isoforms (ZmHXK4-9) was confirmed using a yeast complementation approach. The kinetic parameters of recombinant proteins showed that ZmHXK4-7 have high catalytic efficiency for Glc, fructose (Fru) and mannose (Man), ZmHXK7 has a lower Km for ATP, and together with ZmHXK8 they have lower sensitivity to inhibition by ADP, G6P and N-acetylglucosamine than ZmHXK4-6 and ZmHXK9. Additionally, we demonstrated that ZmHXK4-6 and ZmHXK9 are located in the mitochondria and their location relies on the first 30 amino acids of the N-terminal domain. Otherwise, ZmHXK7-8 are constitutively located in the cytosol. HXK activity was detected in cytosolic and mitochondrial fractions and high Glc and Fru phosphorylating activities were found in imbibed embryos. CONCLUSIONS: Considering the biochemical characteristics, location and the expression of ZmHXK4 at onset of germination, we suggest that it is the main contributor to mitochondrial activity at early germination times, at 24 h other ZmHXKs also contribute to the total activity. While in the cytosol, ZmHXK7 could be responsible for the activity at the onset of germination, although later, ZmHXK8 also contributes to the total HXK activity. Our observations suggest that the HXKs may be redundant proteins with specific roles depending on carbon and ATP availability, metabolic needs, or sensor requirements. Further investigation is necessary to understand their specific or redundant physiological roles.

Mitogen activated protein kinase 6 and MAP kinase phosphatase 1 are involved in the response of Arabidopsis roots to L-glutamate.

KEY MESSAGE: The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.

The MEDIATOR genes MED12 and MED13 control Arabidopsis root system configuration influencing sugar and auxin responses.

KEY MESSAGE: Arabidopsis med12 and med13 mutants exhibit shoot and root phenotypes related to an altered auxin homeostasis. Sucrose supplementation reactivates both cell division and elongation in primary roots as well as auxin-responsive and stem cell niche gene expression in these mutants. An analysis of primary root growth of WT, med12, aux1-7 and med12 aux1 single and double mutants in response to sucrose and/or N-1-naphthylphthalamic acid (NPA) placed MED12 upstream of auxin transport for the sugar modulation of root growth. The MEDIATOR (MED) complex plays diverse functions in plant development, hormone signaling and biotic and abiotic stress tolerance through coordination of transcription. Here, we performed genetic, developmental, molecular and pharmacological analyses to characterize the role of MED12 and MED13 on the configuration of root architecture and its relationship with auxin and sugar responses. Arabidopsis med12 and med13 single mutants exhibit shoot and root phenotypes consistent with altered auxin homeostasis including altered primary root growth, lateral root development, and root hair elongation. MED12 and MED13 were required for activation of cell division and elongation in primary roots, as well as auxin-responsive and stem cell niche gene expression. Remarkably, most of these mutant phenotypes were rescued by supplying sucrose to the growth medium. The growth response of primary roots of WT, med12, aux1-7 and med12 aux1 single and double mutants to sucrose and application of auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) revealed the correlation of med12 phenotype with the activity of the auxin intake permease and suggests that MED12 acts upstream of AUX1 in the root growth response to sugar. These data provide compelling evidence that MEDIATOR links sugar sensing to auxin transport and distribution during root morphogenesis.

Mitogen-Activated Protein Kinase 6 and Ethylene and Auxin Signaling Pathways Are Involved in Arabidopsis Root-System Architecture Alterations by Trichoderma atroviride.

Trichoderma atroviride is a symbiotic fungus that interacts with roots and stimulates plant growth and defense. Here, we show that Arabidopsis seedlings cocultivated with T. atroviride have an altered root architecture and greater biomass compared with axenically grown seedlings. These effects correlate with increased activity of mitogen-activated protein kinase 6 (MPK6). The primary roots of mpk6 mutants showed an enhanced growth inhibition by T. atroviride when compared with wild-type (WT) plants, while T. atroviride increases MPK6 activity in WT roots. It was also found that T. atroviride produces ethylene (ET), which increases with l-methionine supply to the fungal growth medium. Analysis of growth and development of WT seedlings and etr1, ein2, and ein3 ET-related Arabidopsis mutants indicates a role for ET in root responses to the fungus, since etr1 and ein2 mutants show defective root-hair induction and enhanced primary-root growth inhibition when cocultivated with T. atroviride. Increased MPK6 activity was evidenced in roots of ctr1 mutants, which correlated with repression of primary root growth, thus connecting MPK6 signaling with an ET response pathway. Auxin-inducible gene expression analysis using the DR5:uidA reporter construct further revealed that ET affects auxin signaling through the central regulator CTR1 and that fungal-derived compounds, such as indole-3-acetic acid and indole-3-acetaldehyde, induce MPK6 activity. Our results suggest that T. atroviride likely alters root-system architecture modulating MPK6 activity and ET and auxin action.