Large‐scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome‐wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high‐accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high‐confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine‐specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho‐residues in Arabidopsis is similar to that in humans, where over 90 tyrosine‐specific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants.     

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1‐1 and VvLYK1‐2 mediate chitooligosaccharide‐triggered immunity

Chitin, a major component of fungal cell walls, is a well‐known pathogen‐associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM‐RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM‐RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin‐induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1‐1, ‐2, or ‐3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1‐1 and VvLYK1‐2, but not VvLYK1‐3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide‐induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1‐1 in Atcerk1 restored penetration resistance to the non‐adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1‐1 and VvLYK1‐2 participate in chitin‐ and chitosan‐triggered immunity and that VvLYK1‐1 plays an important role in basal resistance against E. necator.     

Ethylene production in Botrytis cinerea‐ and oligogalacturonide‐induced immunity requires calcium‐dependent protein kinases

Plant immunity against pathogens is achieved through rapid activation of defense responses that occur upon sensing of microbe‐ or damage‐associated molecular patterns, respectively referred to as MAMPs and DAMPs. Oligogalacturonides (OGs), linear fragments derived from homogalacturonan hydrolysis by pathogen‐secreted cell wall‐degrading enzymes, and flg22, a 22‐amino acid peptide derived from the bacterial flagellin, represent prototypical DAMPs and MAMPs, respectively. Both types of molecules induce protection against infections. In plants, like in animals, calcium is a second messenger that mediates responses to biotic stresses by activating calcium‐binding proteins. Here we show that simultaneous loss of calcium‐dependent protein kinases CPK5, CPK6 and CPK11 affects Arabidopsis thaliana basal as well as elicitor‐ induced resistance to the necrotroph Botrytis cinerea, by affecting pathogen‐induced ethylene production and accumulation of the ethylene biosynthetic enzymes 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase 2 (ACS2) and 6 (ACS6). Moreover, ethylene signaling contributes to OG‐triggered immunity activation, and lack of CPK5, CPK6 and CPK11 affects the duration of OG‐ and flg22‐induced gene expression, indicating that these kinases are shared elements of both DAMP and MAMP signaling pathways.     

The secret to life is being different: asymmetric divisions in plant development

Asymmetric cell divisions (ACDs) are used to create organismal form and cellular diversity during plant development. In several embryonic and postembryonic contexts, genes that specify cell fates and networks that provide positional information have been identified. The cellular mechanisms that translate this information into a physically ACD, however, are still obscure. In this review we examine the cell polarization events that precede asymmetric divisions in plants. Using principles derived from studies of other organisms and from postmitotic polarity generation in plants, we endeavor to provide a framework of what is known, what is on the horizon and what is critically needed to develop a rigorous mechanistic understanding of ACDs in plants.     

Involvement of NtERF3 in the cell death signalling pathway mediated by SIPK/WIPK and WRKY1 in tobacco plants

We previously reported that one of the ethylene response factors (ERFs), NtERF3, and other members of the subgroup VIII‐a ERFs of the AP2/ERF family exhibit cell death‐inducing ability in tobacco leaves. In this study, we focused on the involvement of NtERF3 in a cell death signalling pathway in tobacco plants, particularly downstream of NtSIPK/NtWIPK and NtWRKY1, which are mitogen‐activated protein kinases and a phosphorylation substrate of NtSIPK, respectively. An ERF‐associated amphiphilic repression (EAR) motif‐deficient NtERF3b mutant (NtERF3bΔEAR) that lacked cell death‐inducing ability suppressed the induction of cell death caused by NtERF3a. The transient co‐expression of NtERF3bΔEAR suppressed the hypersensitive reaction (HR)‐like cell death induced by NtSIPK and NtWRKY1. The induction of cell death by NtSIPK and NtWRKY1 was also inhibited in transgenic plants expressing NtERF3bΔEAR. Analysis of gene expression, ethylene production and cell death symptoms in salicylic acid‐deficient tobacco plants suggested the existence of some feedback regulation in the HR cell death signalling pathway mediated by SIPK/WIPK and WRKY1. Overall, these results suggest that NtERF3 functions downstream of NtSIPK/NtWIPK and NtWRKY1 in a cell death signalling pathway, with some feedback regulation.     

The impact of Arabidopsis thaliana SNF1‐related‐kinase 1 (SnRK1)‐activating kinase 1 (SnAK1) and SnAK2 on SnRK1 phosphorylation status: characterization of a SnAK double mutant

Arabidopsis thaliana SNF1‐related‐kinase 1 (SnRK1)‐activating kinase 1 (AtSnAK1) and AtSnAK2 have been shown to phosphorylate in vitro and activate the energy signalling integrator, SnRK1. To clarify this signalling cascade in planta, a genetic‐ and molecular‐based approach was developed. Homozygous single AtSnAK1 and AtSnAK2 T‐DNA insertional mutants did not display an apparent phenotype. Crossing of the single mutants did not allow the isolation of double‐mutant plants, whereas self‐pollinating the S1?/? S2+/? sesquimutant specifically gave approximatively 22% individuals in their offspring that, when rescued on sugar‐supplemented media in vitro, were shown to be AtSnAK1 AtSnAK2 double mutants. Interestingly, this was not obtained in the case of the other sesquimutant, S1+/? S2?/?. Although reduced in size, the double mutant had the capacity to produce flowers, but not seeds. Immunological characterization established the T‐loop of the SnRK1 catalytic subunit to be non‐phosphorylated in the absence of both SnAKs. When the double mutant was complemented with a DNA construct containing an AtSnAK2 open reading frame driven by its own promoter, a normal phenotype was restored. Therefore, wild‐type plant growth and development is dependent on the presence of SnAK in vivo, and this is correlated with SnRK1 phosphorylation. These data show that both SnAKs are kinases phosphorylating SnRK1, and thereby they contribute to energy signalling in planta.     

Spindle orientation and epidermal morphogenesis

Asymmetric cell divisions (ACDs) result in two unequal daughter cells and are a hallmark of stem cells. ACDs can be achieved either by asymmetric partitioning of proteins and organelles or by asymmetric cell fate acquisition due to the microenvironment in which the daughters are placed. Increasing evidence suggests that in the mammalian epidermis, both of these processes occur. During embryonic epidermal development, changes occur in the orientation of the mitotic spindle in relation to the underlying basement membrane. These changes are guided by conserved molecular machinery that is operative in lower eukaryotes and dictates asymmetric partitioning of proteins during cell divisions. That said, the shift in spindle alignment also determines whether a division will be parallel or perpendicular to the basement membrane, and this in turn provides a differential microenvironment for the resulting daughter cells. Here, we review how oriented divisions of progenitors contribute to the development and stratification of the epidermis.     

Two guard cell mitogen‐activated protein kinases,MPK9 and MPK12, function in methyl jasmonate‐induced stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell‐preferential mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9‐1 and mpk12‐1 single mutants as well as wild‐type plants, but not in mpk9‐1 mpk12‐1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA‐induced stomatal closure in wild‐type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9‐1, mpk12‐1 and mpk9‐1 mpk12‐1 mutants, as well in wild‐type plants. Furthermore, MeJA triggered elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in the mpk9‐1 mpk12‐1 double mutant as well as wild‐type plants. Activation of S‐type anion channels by MeJA was impaired in mpk9‐1 mpk12‐1. Together, these results indicate that MPK9 and MPK12 function upstream of S‐type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca²⁺]_cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.     

Cyclin‐dependent kinase‐activating kinases CDKD;1 and CDKD;3 are essential for preserving mitotic activity in Arabidopsis thaliana

For the full activation of cyclin‐dependent kinases (CDKs), not only cyclin binding but also CDK phosphorylation is required. This activating phosphorylation is mediated by CDK‐activating kinases (CAKs). Arabidopsis has four genes showing similarity to vertebrate‐type CAKs, three CDKDs (CDKD;1‐CDKD;3) and one CDKF (CDKF;1). We previously found that the cdkf;1 mutant is defective in post‐embryonic development, even though the kinase activities of core CDKs remain unchanged relative to the wild type. This raised a question about the involvement of CDKDs in CDK activation in planta. Here we report that the cdkd;1 cdkd;3 double mutant showed gametophytic lethality. Most cdkd;1‐1 cdkd;3‐1 pollen grains were defective in pollen mitosis I and II, producing one‐cell or two‐cell pollen grains that lacked fertilization ability. We also found that the double knock‐out of CDKD;1 and CDKD;3 caused arrest and/or delay in the progression of female gametogenesis at multiple steps. Our genetic analyses revealed that the functions of CDKF;1 and CDKD;1 or CDKD;3 do not overlap, either during gametophyte and embryo development or in post‐embryonic development. Consistent with these analyses, CDKF;1 expression in the cdkd;1‐1 cdkd;3‐1 mutant could not rescue the gametophytic lethality. These results suggest that, in Arabidopsis, CDKD;1 and CDKD;3 function as CAKs controlling mitosis, whereas CDKF;1 plays a distinct role, mainly in post‐embryonic development. We propose that CDKD;1 and CDKD;3 phosphorylate and activate all core CDKs, CDKA, CDKB1 and CDKB2, thereby governing cell cycle progression throughout plant development.     

Mammalian protein histidine kinases

The existence of protein kinases, known as histidine kinases, which phosphorylate their substrates on histidine residues has been well documented in bacteria and also in lower eukaryotes such as yeast and plants. Their biological roles in cellular signalling pathways within these organisms have also been well characterised. The evidence for the existence of such enzymes in mammalian cells is much less well established and little has been determined about their cellular functions. The aim of the current review is to present a summary of what is known about mammalian histidine kinases. In addition, by consideration of the chemistry of phosphohistidine, what is currently known of some mammalian histidine kinases and the way in which they act in bacteria and other eukaryotes, a general role for mammalian histidine kinases is proposed. A histidine kinase phosphorylates a substrate protein, by virtue of the relatively high free energy of hydrolysis of phosphohistidine the phosphate group is easily transferred to either a small molecule or another protein with which the phosphorylated substrate protein specifically interacts. This allows a signalling process to occur, which may be downregulated by the action of phosphatases. Given the known importance of protein phosphorylation to the regulation of almost all aspects of cellular function, the investigation of the largely unexplored area of histidine phosphorylation in mammalian cells is likely to provide a greater understanding of cellular action and possibly provide a new set of therapeutic drug targets.     

A THEMIS:SHP1 complex promotes T‐cell survival

THEMIS is critical for conventional T‐cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr‐phosphorylation‐independent fashion. Rather, SHP1 and THEMIS engage with the N‐SH3 and C‐SH3 domains of GRB2, respectively, a configuration that allows GRB2‐SH2 to recruit the complex onto LAT. Consistent with THEMIS‐mediated recruitment of SHP to the TCR signalosome, THEMIS knock‐down increased TCR‐induced CD3‐ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock‐down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK‐mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T‐cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T‐cell development and differentiation.