Plants make galls to accommodate foreigners: some are friends, most are foes.

At the colonization site of a foreign entity, plant cells alter their trajectory of growth and development. The resulting structure - a plant gall - accommodates various needs of the foreigner, which are phylogenetically diverse: viruses, bacteria, protozoa, oomycetes, true fungi, parasitic plants, and many types of animals, including rotifers, nematodes, insects, and mites. The plant species that make galls also are diverse. We assume gall production costs the plant. All is well if the foreigner provides a gift that makes up for the cost. Nitrogen-fixing nodule-inducing bacteria provide nutritional services. Gall wasps pollinate fig trees. Unfortunately for plants, most galls are made for foes, some of which are deeply studied pathogens and pests: Agrobacterium tumefaciens, Rhodococcus fascians, Xanthomonas citri, Pseudomonas savastanoi, Pantoea agglomerans, 'Candidatus' phytoplasma, rust fungi, Ustilago smuts, root knot and cyst nematodes, and gall midges. Galls are an understudied phenomenon in plant developmental biology. We propose gall inception for discovering unifying features of the galls that plants make for friends and foes, talk about molecules that plants and gall-inducers use to get what they want from each other, raise the question of whether plants colonized by arbuscular mycorrhizal fungi respond in a gall-like manner, and present a research agenda.

Molecular dynamics in germinating, endophyte-colonized quinoa seeds.

AIMS: The pseudo-cereal quinoa has an outstanding nutritional value. Seed germination is unusually fast, and plant tolerance to salt stress exceptionally high. Seemingly all seeds harbor bacterial endophytes. This work examines mitogen-activated protein kinase (MAPK) activities during early development. It evaluates possible contribution of endophytes to rapid germination and plant robustness. METHODS: MAPK activities were monitored in water- and NaCl-imbibed seeds over a 4-h-period using an immunoblot-based approach. Cellulolytic and pectinolytic abilities of bacteria were assessed biochemically, and cellular movement, biofilm, elicitor and antimicrobial compound synthesis genes sequenced. GyrA-based, cultivation-independent studies provided first insight into endophyte diversity. RESULTS: Quinoa seeds and seedlings exhibit remarkably complex and dynamic MAPK activity profiles. Depending on seed origin, variances exist in MAPK patterns and probably also in endophyte assemblages. Mucilage-degrading activities enable endophytes to colonize seed surfaces of a non-host species, chia, without apparent adverse effects. CONCLUSIONS: Owing to their motility, cell wall-loosening and elicitor-generating abilities, quinoa endophytes have the potential to drive cell expansion, move across cell walls, generate damage-associated molecular patterns and activate MAPKs in their host. Bacteria may thus facilitate rapid germination and confer a primed state directly upon seed rehydration. Transfer into non-native crops appears both desirable and feasible.

Developmental Peculiarities and Seed-Borne Endophytes in Quinoa: Omnipresent, Robust Bacilli Contribute to Plant Fitness.

Among potential climate change-adapted crops for future agriculture, quinoa (Chenopodium quinoa), a facultative halophyte plant with exceptional nutritional properties, stands out as a prime candidate. This work examined how quinoa deals with extreme situations during seed rehydration. Quinoa distinguishes itself from other plants in multiple ways. It germinates within minutes, even under extremely hostile conditions. Broken seeds/split embryos are able to regenerate. Furthermore, quinoa seedlings are resurrection-competent. These peculiarities became in part explainable upon discovery of seed-borne microorganisms. 100% of quinoa seeds, from different sources, are inhabited by diverse members of the genus Bacillus. These endophytes are motile and reside in all seedling organs, indicating vertical transmission. Owing to their high catalase activities and superoxide contents the bacteria potentially manipulate the host's redox status. Superoxide-driven cell expansion enables quinoa to overcome a critical period in development, seedling establishment. Quinoa's immediate confrontation with "foreign" reactive oxygen species and bacterial elicitors likely induces a naturally primed state, enabling plants to withstand extreme situations. The endophytic bacteria, which are cultivable and highly robust themselves, have high potential for application in agriculture, food (amylase) and cosmetics (catalase) industry. This work also discusses the potential of transferring quinoa's microbiome to improve stress resistance in other plant species.

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis.

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

Antioxidative responses during germination in quinoa grown in vitamin B-rich medium.

Synthetic vitamin preparations have grown in popularity to combat health risks associated with an imbalanced diet, poor exercise and stress. In terms of bioavailability and diversity, they lack behind vitamins naturally occurring in plants. Solutions to obtain plant-derived vitamins at a larger scale are highly desirable. B vitamins act as precursors of enzymatic cofactors, thereby regulating important metabolic processes both in animals and plants. Because during plant germination, the vitamin content and micronutrient availability increase, sprouts are generally considered a healthier food as compared to dry grains. Germination only occurs if a plant's antioxidant machinery is sufficiently activated to cope with oxidative stress. Seeds of quinoa, an edible gluten-free plant naturally rich in minerals, germinate readily in a solution containing the eight B vitamins. We studied biochemical changes during quinoa germination, with a focus on nutritionally relevant characteristics. The results are considered from a nutritional and plant physiological perspective. Germination of quinoa in vitamin-rich medium is a promising strategy to enhance the nutritional value of this matrix. Additional health-beneficial effects indirectly resulting from the vitamin treatment include elevated levels of the multi-functional amino acid proline and a higher antioxidant capacity. Plant biomolecules can be better protected from oxidative damage in vivo.

Modes of MAPK substrate recognition and control.

Mitogen-activated protein kinase (MAPK) cascades are universal, evolutionary conserved signalling modules, which translate environmental information into appropriate responses via phosphorylation of their substrate proteins. In Arabidopsis, the MAPK MPK3 regulates numerous cellular processes, including the adaptation to abiotic and biotic stresses. The molecular steps immediately downstream of MPK3 induction have, therefore, received abundant attention, and a respectable number of MPK3 targets are known by now. These proteins illustrate the substrate promiscuity of MPK3. They also are evidence for how manifold phosphorylation-regulated functions can be. This review presents the current knowledge about the function and regulation of MPK3-targeted proteins, takes a close look at their primary protein sequences, and proposes a model of how MPK3 recognises, binds, and phosphorylates its targets.

Mitogen-activated protein kinase-regulated AZI1 - an attractive candidate for genetic engineering.

Mitogen-activated protein kinases and their targets have been in the limelight of plant stress research. Signaling pathways mediating the responses to multiple stresses deserve particular attention. In a recent study, we reported AZI1, a member of the lipid transfer protein family, to play a role in MPK3-mediated responses to salt stress in Arabidopsis thaliana. MPK3 controls AZI1 at the transcriptional and posttranslational level. The AZI1 protein has several properties that make it very attractive for genetic engineering. A model of multi-level control of AZI1 by MPK3 is proposed, and strategies toward optimizing AZI1 protein properties are briefly discussed.

Dominant repression by Arabidopsis transcription factor MYB44 causes oxidative damage and hypersensitivity to abiotic stress.

In any living species, stress adaptation is closely linked with major changes of the gene expression profile. As a substrate protein of the rapidly stress-induced mitogen-activated protein kinase MPK3, Arabidopsis transcription factor MYB44 likely acts at the front line of stress-induced re-programming. We recently characterized MYB44 as phosphorylation-dependent positive regulator of salt stress signaling. Molecular events downstream of MYB44 are largely unknown. Although MYB44 binds to the MBSII element in vitro, it has no discernible effect on MBSII-driven reporter gene expression in plant co-transfection assays. This may suggest limited abundance of a synergistic co-regulator. MYB44 carries a putative transcriptional repression (Ethylene responsive element binding factor-associated Amphiphilic Repression, EAR) motif. We employed a dominant repressor strategy to gain insights into MYB44-conferred stress resistance. Overexpression of a MYB44-REP fusion markedly compromised salt and drought stress tolerance--the opposite was seen in MYB44 overexpression lines. MYB44-mediated resistance likely results from induction of tolerance-enhancing, rather than from repression of tolerance-diminishing factors. Salt stress-induced accumulation of destructive reactive oxygen species is efficiently prevented in transgenic MYB44, but accelerated in MYB44-REP lines. Furthermore, heterologous overexpression of MYB44-REP caused tissue collapse in Nicotiana. A mechanistic model of MAPK-MYB-mediated enhancement in the antioxidative capacity and stress tolerance is proposed. Genetic engineering of MYB44 variants with higher trans-activating capacity may be a means to further raise stress resistance in crops.

Salt stress in Arabidopsis: lipid transfer protein AZI1 and its control by mitogen-activated protein kinase MPK3.

A plant's capability to cope with environmental challenges largely relies on signal transmission through mitogen-activated protein kinase (MAPK) cascades. In Arabidopsis thaliana, MPK3 is particularly strongly associated with numerous abiotic and biotic stress responses. Identification of MPK3 substrates is a milestone towards improving stress resistance in plants. Here, we characterize AZI1, a lipid transfer protein (LTP)-related hybrid proline-rich protein (HyPRP), as a novel target of MPK3. AZI1 is phosphorylated by MPK3 in vitro. As documented by co-immunoprecipitation and bimolecular fluorescence complementation experiments, AZI1 interacts with MPK3 to form protein complexes in planta. Furthermore, null mutants of azi1 are hypersensitive to salt stress, while AZI1-overexpressing lines are markedly more tolerant. AZI1 overexpression in the mpk3 genetic background partially alleviates the salt-hypersensitive phenotype of this mutant, but functional MPK3 appears to be required for the full extent of AZI1-conferred robustness. Notably, this robustness does not come at the expense of normal development. Immunoblot and RT-PCR data point to a role of MPK3 as positive regulator of AZI1 abundance.

Tropaeolum tops tobacco - simple and efficient transgene expression in the order Brassicales.

Transient expression systems are valuable tools in molecular biology. Agrobacterial infiltration of leaves is well-established in tobacco, but has led to limited success in the model plant Arabidopsis thaliana. An efficient expression system combining the advantages of Arabidopsis (well-characterised) and the simplicity of leaf infiltration is desirable. Here, I describe Agrobacterium tumefaciens-mediated transformation of Tropaeolummajus (nasturtium, order Brassicales) as a remarkably simple, cheap and highly efficient transient expression system. It provides the Arabidopsis community with a tool to study subcellular localisation, protein-protein interactions and reporter gene activities (e.g. luciferase, ß-glucuronidase) in a genetic background that is closely related to their primary model organism. Unlike Arabidopsis, Tropaeolum is capable of engaging in endomycorrhizal associations and is therefore relevant also to symbiosis research. RNAi-based approaches are more likely to succeed than in the distantly-related Nicotiana transformation system. Tropaeolummajus was voted the "medicinal plant of the year 2013". Conquering this plant for genetic manipulations harbours potential for biotechnological and pharmacological applications.

Tight interconnection and multi-level control of Arabidopsis MYB44 in MAPK cascade signalling.

Abiotic stress poses a huge, ever-increasing problem to plants and agriculture. The dissection of signalling pathways mediating stress tolerance is a prerequisite to develop more resistant plant species. Mitogen-activated protein kinase (MAPK) cascades are universal signalling modules. In Arabidopsis, the MAPK MPK3 and its upstream regulator MAPK kinase MKK4 initiate the adaptation response to numerous abiotic and biotic stresses. Yet, molecular steps directly linked with MKK4-MPK3 activation are largely unknown. Starting with a yeast-two-hybrid screen for interacting partners of MKK4, we identified a transcription factor, MYB44. MYB44 is controlled at multiple levels by and strongly inter-connected with MAPK signalling. As we had shown earlier, stress-induced expression of the MYB44 gene is regulated by a MPK3-targeted bZIP transcription factor VIP1. At the protein level, MYB44 interacts with MPK3 in vivo. MYB44 is phosphorylated by MPK3 in vitro at a single residue, Ser145. Although replacement of Ser145 by a non-phosphorylatable (S145A) or phosphomimetic (S145D) residue did not alter MYB44 subcellular localisation, dimerization behaviour nor DNA-binding characteristics, abiotic stress tolerance tests in stable transgenic Arabidopsis plants clearly related S145 phosphorylation to MYB44 function: Compared to Arabidopsis wild type plants, MYB44 overexpressing lines exhibit an enhanced tolerance to osmotic stress and are slightly more sensitive to abscisic acid. Interestingly, overexpression of the S145A variant revealed that impaired phosphorylation does not render the MYB44 protein non-functional. Instead, S145A lines are highly sensitive to abiotic stress, and thereby remarkably similar to mpk3-deficient plants. Its in vivo interaction with the nuclear sub-pools of both MPK3 and MKK4 renders MYB44 the first plant transcription factor to have a second function as putative MAPK cascade scaffolding protein.

Brassinosteroid-regulated GSK3/Shaggy-like kinases phosphorylate mitogen-activated protein (MAP) kinase kinases, which control stomata development in Arabidopsis thaliana.

Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module.

Agrobacterium infection and plant defense-transformation success hangs by a thread.

The value of Agrobacterium tumefaciens for plant molecular biologists cannot be appreciated enough. This soil-borne pathogen has the unique capability to transfer DNA (T-DNA) into plant systems. Gene transfer involves both bacterial and host factors, and it is the orchestration of these factors that determines the success of transformation. Some plant species readily accept integration of foreign DNA, while others are recalcitrant. The timing and intensity of the microbially activated host defense repertoire sets the switch to "yes" or "no." This repertoire is comprised of the specific induction of mitogen-activated protein kinases (MAPKs), defense gene expression, production of reactive oxygen species (ROS) and hormonal adjustments. Agrobacterium tumefaciens abuses components of the host immunity system it mimics plant protein functions and manipulates hormone levels to bypass or override plant defenses. A better understanding of the ongoing molecular battle between agrobacteria and attacked hosts paves the way toward developing transformation protocols for recalcitrant plant species. This review highlights recent findings in agrobacterial transformation research conducted in diverse plant species. Efficiency-limiting factors, both of plant and bacterial origin, are summarized and discussed in a thought-provoking manner.

Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies.

BACKGROUND: Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to few plant species often requiring substantial equipment and facilities. High chloroplast and chlorophyll content may further impede downstream applications of transformed cells from green plant tissue. RESULTS: Here, we describe a fast and simple technique for the high-yield isolation and efficient transformation (>70%) of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima). In this method no particular growth facilities or expensive equipments are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in a variety of commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence) or promoter activity studies. Due to low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence) are alleviated. Furthermore, the transgene expression is detectable within 90 minutes of transformation and lasts for several days. CONCLUSIONS: The simplicity of the isolation and transformation procedure renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multi-colour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications.

New insights into an old story: Agrobacterium-induced tumour formation in plants by plant transformation.

Agrobacterium tumefaciens causes tumour formation in plants. Plant signals induce in the bacteria the expression of a range of virulence (Vir) proteins and the formation of a type IV secretion system (T4SS). On attachment to plant cells, a transfer DNA (T-DNA) and Vir proteins are imported into the host cells through the bacterial T4SS. Through interaction with a number of host proteins, the Vir proteins suppress the host innate immune system and support the transfer, nuclear targeting, and integration of T-DNA into host cell chromosomes. Owing to extensive genetic analyses, the bacterial side of the plant-Agrobacterium interaction is well understood. However, progress on the plant side has only been achieved recently, revealing a highly complex molecular choreography under the direction of the Vir proteins that impinge on multiple processes including transport, transcription, and chromosome status of their host cells.

VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression.

The plant pathogen Agrobacterium tumefaciens transforms plant cells by delivering its T-DNA into the plant cell nucleus where it integrates into the plant genome and causes tumor formation. A key role of VirE2-interacting protein 1 (VIP1) in the nuclear import of T-DNA during Agrobacterium-mediated plant transformation has been unravelled and VIP1 was shown to undergo nuclear localization upon phosphorylation by the mitogen-activated protein kinase MPK3. Here, we provide evidence that VIP1 encodes a functional bZIP transcription factor that stimulates stress-dependent gene expression by binding to VIP1 response elements (VREs), a DNA hexamer motif. VREs are overrepresented in promoters responding to activation of the MPK3 pathway such as Trxh8 and MYB44. Accordingly, plants overexpressing VIP1 accumulate high levels of Trxh8 and MYB44 transcripts, whereas stress-induced expression of these genes is impaired in mpk3 mutants. Trxh8 and MYB44 promoters are activated by VIP1 in a VRE-dependent manner. VIP1 strongly enhances expression from a synthetic promoter harboring multiple VRE copies and directly interacts with VREs in vitro and in vivo. Chromatin immunoprecipitation assays of the MYB44 promoter confirm that VIP1 binding to VREs is enhanced under conditions of MPK3 pathway stimulation. These results provide molecular insight into the cellular mechanism of target gene regulation by the MPK3 pathway.

MAPK cascade signalling networks in plant defence.

The sensing of stress signals and their transduction into appropriate responses is crucial for the adaptation and survival of plants. Kinase cascades of the mitogen-activated protein kinase (MAPK) class play a remarkably important role in plant signalling of a variety of abiotic and biotic stresses. MAPK cascade-mediated signalling is an essential step in the establishment of resistance to pathogens. Here, we describe the most recent insights into MAPK-mediated pathogen defence response regulation with a particular focus on the cascades involving MPK3, MPK4 and MPK6. We also discuss the strategies developed by plant pathogens to circumvent, inactivate or even 'hijack' MAPK-mediated defence responses.

A major role of the MEKK1-MKK1/2-MPK4 pathway in ROS signalling.

Over the last few years, it has become evident that reactive oxygen species (ROS) signalling plays an important role in various physiological responses, including pathogen defense and stomatal opening/closure. On the other hand, ROS overproduction is detrimental for proper plant growth and development, indicating that the regulation of an appropriate redox balance is essential for plants. ROS homeostasis in plants involves the mitogen-activated protein kinase (MAPK) pathway consisting of the MAPK kinase kinase MEKK1 and the MAPK MPK4. Phenotypic and molecular analysis revealed that the MAPK kinases MKK1 and MKK2 are part of a cascade, regulating ROS and salicylic acid (SA) accumulation. Gene expression analysis shows that of 32 transcription factors reported to be highly responsive to multiple ROS-inducing conditions, 20 are regulated by the MEKK1, predominantly via the MEKK1-MKK1/2-MPK4 pathway. However, MEKK1 also functions on other as yet unknown pathways and part of the MEKK1-dependent MPK4 responses are regulated independently of MKK1 and MKK2. Overall, this analysis emphasizes the central role of this MAPK cascade in oxidative stress signalling, but also indicates the high level of complexity revealed by this signalling network.