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.

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.

Functional analysis of the Arabidopsis thaliana CHLOROPLAST BIOGENESIS 19 pentatricopeptide repeat editing protein.

The Arabidopsis thaliana pentatricopeptide repeat (PPR) family of proteins contains several degenerate 35-aa motifs named PPR repeats. These proteins control diverse post-transcriptional regulatory mechanisms, including RNA editing. CLB19 belongs to the PLS subfamily of PPR proteins and is essential for the editing and functionality of the subunit A of plastid-encoded RNA polymerase (RpoA) and the catalytic subunit of the Clp protease (ClpP1). We demonstrate in vitro that CLB19 has a specific interaction with these two targets, in spite of their modest sequence similarity. Using site-directed mutagenesis of the rpoA target, we analyzed the essential nucleotides required for CLB19-rpoA interactions. We verified that, similar to other editing proteins, the C-terminal E domain of CLB19 is essential for editing but not for RNA binding. Using biomolecular fluorescence complementation, we demonstrated that the E domain of CLB19 interacts with the RNA-interacting protein MORF2/RIP2 but not with MORF9/RIP9. An interesting finding from this analysis was that overexpression of a truncated CLB19 protein lacking the E domain interferes with cell fate during megasporogenesis and the subsequent establishment of a female gametophyte, supporting an important role of plastids in female gametogenesis. Together these analyses provide important clues about the particularities of the CLB19 editing protein.

Sugar regulation of SUGAR TRANSPORTER PROTEIN 1 (STP1) expression in Arabidopsis thaliana.

Sugars regulate the expression of many genes at the transcriptional level. In Arabidopsis thaliana, sugars induce or repress the expression of >1800 genes, including the STP1 (SUGAR TRANSPORTER PROTEIN 1) gene, which encodes an H(+)/monosaccharide cotransporter. STP1 transcript levels decrease more rapidly after the addition of low concentrations of sugars than the levels of other repressed genes, such as DIN6 (DARK-INDUCED 6). We found that this regulation is exerted at the transcriptional level and is initiated by phosphorylatable sugars. Interestingly, the sugar signal that modulates STP1 expression is transmitted through a HEXOKINASE 1-independent signalling pathway. Finally, analysis of the STP1 5' regulatory region allowed us to delimit a region of 309bp that contains the cis elements implicated in the glucose regulation of STP1 expression. Putative cis-acting elements involved in this response were identified.

An Uncharacterized Apocarotenoid-Derived Signal Generated in ζ-Carotene Desaturase Mutants Regulates Leaf Development and the Expression of Chloroplast and Nuclear Genes in Arabidopsis.

In addition to acting as photoprotective compounds, carotenoids also serve as precursors in the biosynthesis of several phytohormones and proposed regulatory signals. Here, we report a signaling process derived from carotenoids that regulates early chloroplast and leaf development. Biosynthesis of the signal depends on ζ-carotene desaturase activity encoded by the ζ-CAROTENE DESATURASE (ZDS)/CHLOROPLAST BIOGENESIS5 (CLB5) gene in Arabidopsis thaliana. Unlike other carotenoid-deficient plants, zds/clb5 mutant alleles display profound alterations in leaf morphology and cellular differentiation as well as altered expression of many plastid- and nucleus-encoded genes. The leaf developmental phenotypes and gene expression alterations of zds/clb5/spc1/pde181 plants are rescued by inhibitors or mutations of phytoene desaturase, demonstrating that phytofluene and/or ζ-carotene are substrates for an unidentified signaling molecule. Our work further demonstrates that this signal is an apocarotenoid whose synthesis requires the activity of the carotenoid cleavage dioxygenase CCD4.

MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis.

Long chain bases (LCBs) are sphingolipid intermediates acting as second messengers in programmed cell death (PCD) in plants. Most of the molecular and cellular features of this signaling function remain unknown. We induced PCD conditions in Arabidopsis thaliana seedlings and analyzed LCB accumulation kinetics, cell ultrastructure and phenotypes in serine palmitoyltransferase (spt), mitogen-activated protein kinase (mpk), mitogen-activated protein phosphatase (mkp1) and lcb-hydroxylase (sbh) mutants. The lcb2a-1 mutant was unable to mount an effective PCD in response to fumonisin B1 (FB1), revealing that the LCB2a gene is essential for the induction of PCD. The accumulation kinetics of LCBs in wild-type (WT) and lcb2a-1 plants and reconstitution experiments with sphinganine indicated that this LCB was primarily responsible for PCD elicitation. The resistance of the null mpk6 mutant to manifest PCD on FB1 and sphinganine addition and the failure to show resistance on pathogen infection and MPK6 activation by FB1 and LCBs indicated that MPK6 mediates PCD downstream of LCBs. This work describes MPK6 as a novel transducer in the pathway leading to LCB-induced PCD in Arabidopsis, and reveals that sphinganine and the LCB2a gene are required in a PCD process that operates as one of the more effective strategies used as defense against pathogens in plants.

CLB19, a pentatricopeptide repeat protein required for editing of rpoA and clpP chloroplast transcripts.

RNA editing changes the sequence of many transcripts in plant organelles, but little is known about the molecular mechanisms determining the specificity of the process. In this study, we have characterized CLB19 (also known as PDE247), a gene that is required for editing of two distinct chloroplast transcripts, rpoA and clpP. Loss-of-function clb19 mutants present a yellow phenotype with impaired chloroplast development and early seedling lethality under greenhouse conditions. Transcript patterns are profoundly affected in the mutant plants, with a pattern entirely consistent with a defect in activity of the plastid-encoded RNA polymerase. CLB19 encodes a pentatricopeptide repeat protein similar to the editing specificity factors CRR4 and CRR21, but, unlike them, is implicated in editing of two target sites.