Melatonin functions in priming of stomatal immunity in Panax notoginseng and Arabidopsis thaliana

Melatonin (MT) plays important roles in plant disease response, but the mechanisms are largely unknown. Here, we show that MT functions in stomatal immunity in Panax notoginseng and Arabidopsis thaliana. Biochemical analyses showed that MT-induced stomatal closure plays a prominent role in preventing invasion of bacteria Pseudomonas syringe pv. tomato (Pst) DC3000 via activation of mitogen-activated protein kinase (MAPK) and NADPH oxidase-mediated reactive oxygen species production in P. notoginseng. The first putative phytomelatonin receptor 1 (PMTR1) is a plasma membrane protein required for perceiving MT signaling in stomatal closure and activation of MAPK. Biochemical and genetic tests found PMTR1 is essential for flg22- and MT-induced MAPK activation in a heterotrimeric GTP-binding protein Gα subunit GPA1-independent manner. GPA1 functions in the same genetic pathways of FLS2/BAK1 (Flagellin Sensing 2/Brassinosteroid Insensitive 1-associated kinase 1)- as well as PMTR1-mediated flg22 and MT signaling in stomatal closure. The stomata in pmtr1 are insensitive to MT and flg22, but the application of MT induces stomatal closure and reduces the bacterial growth in fls2 and bak1 plants, indicating that PMTR1 might be a downstream signaling component in FLS2- and BAK1-mediated stomatal immunity. In summary, our results (i) demonstrate that phytomelatonin functions in the priming of stomatal immunity and (ii) provide insights into the phytomelatonin signaling transduction pathway.

Melatonin regulates plant innate immunity via the putative receptor PMTR1, which mediates the activation of MAPK cascades and GPA1 signaling.     

Calcium-dependent protein kinase 29 modulates PIN-FORMED polarity and Arabidopsis development via its own phosphorylation code

PIN-FORMED (PIN)-mediated polar auxin transport (PAT) is involved in key developmental processes in plants. Various internal and external cues influence plant development via the modulation of intracellular PIN polarity and, thus, the direction of PAT, but the mechanisms underlying these processes remain largely unknown. PIN proteins harbor a hydrophilic loop (HL) that has important regulatory functions; here, we used the HL as bait in protein pulldown screening for modulators of intracellular PIN trafficking in Arabidopsis thaliana. Calcium-dependent protein kinase 29 (CPK29), a Ca²⁺-dependent protein kinase, was identified and shown to phosphorylate specific target residues on the PIN-HL that were not phosphorylated by other kinases. Furthermore, loss of CPK29 or mutations of the phospho-target residues in PIN-HLs significantly compromised intracellular PIN trafficking and polarity, causing defects in PIN-mediated auxin redistribution and biological processes such as lateral root formation, root twisting, hypocotyl gravitropism, phyllotaxis, and reproductive development. These findings indicate that CPK29 directly interprets Ca²⁺ signals from internal and external triggers, resulting in the modulation of PIN trafficking and auxin responses.

Ca²⁺-dependent protein kinase 29 directly phosphorylates the hydrophilic loop of PIN-FORMED proteins to modulate their intracellular trafficking and Arabidopsis development.     

Integrated analysis of co-expressed MAP kinase substrates in Arabidopsis thaliana

MAP kinase (MAPK) signal transduction cascades are conserved eukaryotic pathways that modulate stress responses and developmental processes. In a recent report we have identified novel Arabidopsis MAPKK/MAPK/Substrate signaling pathways using microarrays containing 2,158 unique Arabidopsis proteins. Subsequently, several WRKY and TGA targets phosphorylated by MAPKs were verified in planta. We have also reported that specific MAPKK/MAPK modules expressed in Nicotiana benthamiana induced a cell death phenotype related to the immune response. We have generated a MAPK phosphorylation network based on our protein microarray experimental data. Here we further analyze our network by integrating phosphorylation and gene expression information to identify biologically relevant signaling modules. We have identified 108 phosphorylation events that occur among 96 annotated genes with highly similar pairwise expression profiles. Our analysis brings a new perspective on MAPK signaling by revealing new relationships between components of signaling pathways.Key words: MAPK, protein microarray, network, cell death, co-expression, signaling     

Natural variation in stomatal abundance of Arabidopsis thaliana includes cryptic diversity for different developmental processes

Background and Aims

Current understanding of stomatal development in Arabidopsis thaliana is based on mutations producing aberrant, often lethal phenotypes. The aim was to discover if naturally occurring viable phenotypes would be useful for studying stomatal development in a species that enables further molecular analysis.

Methods

Natural variation in stomatal abundance of A. thaliana was explored in two collections comprising 62 wild accessions by surveying adaxial epidermal cell-type proportion (stomatal index) and density (stomatal and pavement cell density) traits in cotyledons and first leaves. Organ size variation was studied in a subset of accessions. For all traits, maternal effects derived from different laboratory environments were evaluated. In four selected accessions, distinct stomatal initiation processes were quantitatively analysed.

Key Results and Conclusions

Substantial genetic variation was found for all six stomatal abundance-related traits, which were weakly or not affected by laboratory maternal environments. Correlation analyses revealed overall relationships among all traits. Within each organ, stomatal density highly correlated with the other traits, suggesting common genetic bases. Each trait correlated between organs, supporting supra-organ control of stomatal abundance. Clustering analyses identified accessions with uncommon phenotypic patterns, suggesting differences among genetic programmes controlling the various traits. Variation was also found in organ size, which negatively correlated with cell densities in both organs and with stomatal index in the cotyledon. Relative proportions of primary and satellite lineages varied among the accessions analysed, indicating that distinct developmental components contribute to natural diversity in stomatal abundance. Accessions with similar stomatal indices showed different lineage class ratios, revealing hidden developmental phenotypes and showing that genetic determinants of primary and satellite lineage initiation combine in several ways. This first systematic, comprehensive natural variation survey for stomatal abundance in A. thaliana reveals cryptic developmental genetic variation, and provides relevant relationships amongst stomatal traits and extreme or uncommon accessions as resources for the genetic dissection of stomatal development.     

Architectural strategies of Cornus sericea, a native but invasive shrub of Southern Quebec, Canada, under an open or a closed canopy

Background and Aims

Qualitative and quantitative studies of the pattern of invasive plant development is considered a key aspect in understanding invasiveness. An architectural analysis was therefore performed in order to understand the relationship between shoot architecture and invasiveness in red-osier dogwood, Cornus sericea (Cornaceae).

Methods

The structural and ontogenic characteristics of individuals in invading and non-invading populations in the native range of the species were compared to test the implication of developmental plasticity on invasiveness.

Key Results and Conclusions

The results show that the shrub has a modular architecture governed by strong developmental rules. Cornus sericea is made up of two levels of organization, each with its own intrinsic sequence of differentiation. These intrinsic mechanisms were used as a framework for comparison and it was found that, in response to the light environment, developmental plasticity was elevated, resulting in two architectural strategies. This developmental plasticity concerns the growth direction and the size of the modules, the speed of their time-course changes, their branching and flowering. Under an open canopy, C. sericea rapidly develops large vertical structures and abundant flowering. This strategy leads the plant to be invasive by excluding competitors and disseminating in the landscape. In the understorey, C. sericea slowly develops long horizontal structures which creep across the soil surface, while assimilating structures are poorly developed. This strategy does not lead to invasiveness but may allow the plant to survive in the understorey and reach sunny patches.     

Novel and Expanded Roles for MAPK Signaling in Arabidopsis Stomatal Cell Fate Revealed by Cell Type–Specific Manipulations

Mitogen-activated protein kinase (MAPK) signaling networks regulate numerous eukaryotic biological processes. In Arabidopsis thaliana, signaling networks that contain MAPK kinases MKK4/5 and MAPKs MPK3/6 function in abiotic and biotic stress responses and regulate embryonic and stomatal development. However, how single MAPK modules direct specific output signals without cross-activating additional downstream processes is largely unknown. Studying relationships between MAPK components and downstream signaling outcomes is difficult because broad experimental manipulation of these networks is often lethal or associated with multiple phenotypes. Stomatal development in Arabidopsis follows a series of discrete, stereotyped divisions and cell state transitions. By expressing a panel of constitutively active MAPK kinase (MAPKK) variants in discrete stomatal lineage cell types, we identified a new inhibitory function of MKK4 and MKK5 in meristemoid self-renewal divisions. Furthermore, we established roles for MKK7 and MKK9 as both negative and (unexpectedly) positive regulators during the major stages of stomatal development. This has expanded the number of known MAPKKs that regulate stomatal development and allowed us to build plausible and testable subnetworks of signals. This in vivo cell type–specific assay can be adapted to study other protein families and thus may reveal insights into other complex signal transduction pathways in plants.     

Genome-wide comparative analysis of the <Emphasis Type="Italic">IQD</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Background  

Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues. Stimulus-specific generation of intracellular calcium transients, decoding of calcium signatures, and transformation of the signal into cellular responses are integral modules of the transduction process. Several hundred proteins with functions in calcium signaling circuits have been identified, and the number of downstream targets of calcium sensors is expected to increase. We previously identified a novel, calmodulin-binding nuclear protein, IQD1, which stimulates glucosinolate accumulation and plant defense in Arabidopsis thaliana. Here, we present a comparative genome-wide analysis of a new class of putative calmodulin target proteins in Arabidopsis and rice.     

SAUR proteins and PP2C.D phosphatases regulate H+-ATPases and K+ channels to control stomatal movements

Activation of plasma membrane (PM) H⁺-ATPase activity is crucial in guard cells to promote light-stimulated stomatal opening, and in growing organs to promote cell expansion. In growing organs, SMALL AUXIN UP RNA (SAUR) proteins inhibit the PP2C.D2, PP2C.D5, and PP2C.D6 (PP2C.D2/5/6) phosphatases, thereby preventing dephosphorylation of the penultimate phosphothreonine of PM H⁺-ATPases and trapping them in the activated state to promote cell expansion. To elucidate whether SAUR–PP2C.D regulatory modules also affect reversible cell expansion, we examined stomatal apertures and conductances of Arabidopsis thaliana plants with altered SAUR or PP2C.D activity. Here, we report that the pp2c.d2/5/6 triple knockout mutant plants and plant lines overexpressing SAUR fusion proteins exhibit enhanced stomatal apertures and conductances. Reciprocally, saur56 saur60 double mutants, lacking two SAUR genes normally expressed in guard cells, displayed reduced apertures and conductances, as did plants overexpressing PP2C.D5. Although altered PM H⁺-ATPase activity contributes to these stomatal phenotypes, voltage clamp analysis showed significant changes also in K⁺ channel gating in lines with altered SAUR and PP2C.D function. Together, our findings demonstrate that SAUR and PP2C.D proteins act antagonistically to facilitate stomatal movements through a concerted targeting of both ATP-dependent H⁺ pumping and channel-mediated K⁺ transport.

SMALL AUXIN UP RNA (SAUR) proteins and PP2C.D phosphatases antagonistically regulate stomatal aperture in Arabidopsis by modulating the activities of plasma membrane H⁺-ATPases and K⁺ channels.     

ABA transport factors found in Arabidopsis ABC transporters

Abscisic acid (ABA) is a phytohormone that plays an important role in responses to environmental stresses as well as seed maturation and germination. Intracellular signaling by ABA has been rigorously investigated in relation to stomatal guard-cell regulation, seed germination and abiotic stress responses. However, intercellular regulation of ABA, including the molecular basis of ABA transport systems, has hardly been examined in any plant species. Based on genetic and biochemical analyses, we present evidence that one of the ATP-binding cassette (ABC) transporter genes, AtABCG25, encodes a protein that functions as an ABA exporter through the plasma membrane and is involved in the intercellular ABA signaling pathway. The ABC-type transporter is conserved in model species from E. coli to humans and is reported to transport various metabolites or signaling molecules in an ATP-dependent manner. At same time, another ABC transporter in Arabidopsis, AtABCG40, was independently reported to function as an ABA importer in plant cells. These findings strongly suggest the active control of ABA transport between plant cells, and they provide a novel impetus for examining ABA intercellular regulation.Key words: Arabidopsis, ABA, transport, ABC transporter, ABCG, transposontagged lines     

Lectin receptor-like kinase LecRK-VIII.2 is a missing link in MAPK signaling-mediated yield control

The energy allocation for vegetative and reproductive growth is regulated by developmental signals and environmental cues, which subsequently affects seed output. However, the molecular mechanism underlying how plants coordinate yield-related traits to control yield in changing source–sink relationships remains largely unknown. Here, we discovered the lectin receptor-like kinase LecRK-VIII.2 as a specific receptor-like kinase that coordinates silique number, seed size, and seed number to determine seed yield in Arabidopsis (Arabidopsis thaliana). The lecrk-VIII.2 mutants develop smaller seeds, but more siliques and seeds, leading to increased yield. In contrast, the plants overexpressing LecRK-VIII.2 form bigger seeds, but less siliques and seeds, which results in similar yield to that of wild-type plants. Interestingly, LecRK-VIII.2 promotes the growth of the rosette, root, and stem by coordinating the source–sink relationship. Additionally, LecRK-VIII.2 positively regulates cell expansion and proliferation in the seed coat, and maternally controls seed size. The genetic and biochemical analyses demonstrated that LecRK-VIII.2 acts upstream of the mitogen-activated protein kinase (MAPK) gene MPK6 to regulate silique number, seed size, and seed number. Collectively, these findings uncover LecRK-VIII.2 as an upstream component of the MAPK signaling pathway to control yield-related traits and suggest its potential for crop improvement aimed at developing plants with stable yield, a robust root system, and improved lodging resistance.

A lectin receptor-like kinase regulates yield-related traits and coordinates the source–sink relationship in Arabidopsis.     

Ultrastructure of stomatal development in early-divergent angiosperms reveals contrasting patterning and pre-patterning

Background and Aims

Angiosperm stomata consistently possess a pair of guard cells, but differ between taxa in the patterning and developmental origin of neighbour cells. Developmental studies of phylogenetically pivotal taxa are essential as comparative yardsticks for understanding the evolution of stomatal development.

Methods

We present a novel ultrastructural study of developing stomata in leaves of Amborella (Amborellales), Nymphaea and Cabomba (Nymphaeales), and Austrobaileya and Schisandra (Austrobaileyales), representing the three earliest-divergent lineages of extant angiosperms (the ANITA-grade).

Key Results

Alternative developmental pathways occur in early-divergent angiosperms, resulting partly from differences in pre-patterning and partly from the presence or absence of highly polarized (asymmetric) mitoses in the stomatal cell lineage. Amplifying divisions are absent from ANITA-grade taxa, indicating that ostensible similarities with the stomatal patterning of Arabidopsis are superficial. In Amborella, ‘squared’ pre-patterning occurs in intercostal regions, with groups of four protodermal cells typically arranged in a rectangle; most guard-mother cells are formed by asymmetric division of a precursor cell (the mesoperigenous condition) and are typically triangular or trapezoidal. In contrast, water-lily stomata are always perigenous (lacking asymmetric divisions). Austrobaileya has occasional ‘giant’ stomata.

Conclusions

Similar mature stomatal phenotypes can result from contrasting morphogenetic factors, although the results suggest that paracytic stomata are invariably the product of at least one asymmetric division. Loss of asymmetric divisions in stomatal development could be a significant factor in land plant evolution, with implications for the diversity of key structural and physiological pathways.     

Arabidopsis Raf‐like kinases act as positive regulators of subclass III SnRK2 in osmostress signaling

Given their sessile nature, land plants must use various mechanisms to manage dehydration under water‐deficit conditions. Osmostress‐induced activation of the SNF1‐related protein kinase 2 (SnRK2) family elicits physiological responses such as stomatal closure to protect plants during drought conditions. With the plant hormone ABA receptors [PYR (pyrabactin resistance)/PYL (pyrabactin resistance‐like)/RCAR (regulatory component of ABA receptors) proteins] and group A protein phosphatases, subclass III SnRK2 also constitutes a core signaling module for ABA, and osmostress triggers ABA accumulation. How SnRK2 is activated through ABA has been clarified, although its activation through osmostress remains unclear. Here, we show that Arabidopsis ABA and abiotic stress‐responsive Raf‐like kinases (AtARKs) of the B3 clade of the mitogen‐activated kinase kinase kinase (MAPKKK) family are crucial in SnRK2‐mediated osmostress responses. Disruption of AtARKs in Arabidopsis results in increased water loss from detached leaves because of impaired stomatal closure in response to osmostress. Our findings obtained in vitro and in planta have shown that AtARKs interact physically with SRK2E, a core factor for stomatal closure in response to drought. Furthermore, we show that AtARK phosphorylates S171 and S175 in the activation loop of SRK2E in vitro and that Atark mutants have defects in osmostress‐induced subclass III SnRK2 activity. Our findings identify a specific type of B3‐MAPKKKs as upstream kinases of subclass III SnRK2 in Arabidopsis. Taken together with earlier reports that ARK is an upstream kinase of SnRK2 in moss, an existing member of a basal land plant lineage, we propose that ARK/SnRK2 module is evolutionarily conserved across 400 million years of land plant evolution for conferring protection against drought.     

The emerging role of GABA as a transport regulator and physiological signal

While the proposal that γ-aminobutyric acid (GABA) acts a signal in plants is decades old, a signaling mode of action for plant GABA has been unveiled only relatively recently. Here, we review the recent research that demonstrates how GABA regulates anion transport through aluminum-activated malate transporters (ALMTs) and speculation that GABA also targets other proteins. The ALMT family of anion channels modulates multiple physiological processes in plants, with many members still to be characterized, opening up the possibility that GABA has broad regulatory roles in plants. We focus on the role of GABA in regulating pollen tube growth and stomatal pore aperture, and we speculate on its role in long-distance signaling and how it might be involved in cross talk with hormonal signals. We show that in barley (Hordeum vulgare), guard cell opening is regulated by GABA, as it is in Arabidopsis (Arabidopsis thaliana), to regulate water use efficiency, which impacts drought tolerance. We also discuss the links between glutamate and GABA in generating signals in plants, particularly related to pollen tube growth, wounding, and long-distance electrical signaling, and explore potential interactions of GABA signals with hormones, such as abscisic acid, jasmonic acid, and ethylene. We conclude by postulating that GABA encodes a signal that links plant primary metabolism to physiological status to fine tune plant responses to the environment.

γ-Aminobutyric acid (GABA) encodes a plant signal that links primary metabolism to physiological status to fine tune plant responses to the environment.