A point mutation in the kinase domain of CRK10 leads to xylem vessel collapse and activation of defence responses in Arabidopsis.

Cysteine-rich receptor-like kinases (CRKs) are a large family of plasma membrane-bound receptors ubiquitous in higher plants. However, despite their prominence, their biological roles have remained largely elusive so far. In this study we report the characterization of an Arabidopsis mutant named crk10-A397T in which alanine 397 has been replaced by a threonine in the αC helix of the kinase domain of CRK10, known to be a crucial regulatory module in mammalian kinases. The crk10-A397T mutant is a dwarf that displays collapsed xylem vessels in the root and hypocotyl, whereas the vasculature of the inflorescence develops normally. In situ phosphorylation assays with His-tagged wild type and crk10-A397T versions of the CRK10 kinase domain revealed that both alleles are active kinases capable of autophosphorylation, with the newly introduced threonine acting as an additional phosphorylation site in crk10-A397T. Transcriptomic analysis of wild type and crk10-A397T mutant hypocotyls revealed that biotic and abiotic stress-responsive genes are constitutively up-regulated in the mutant, and a root-infection assay with the vascular pathogen Fusarium oxysporum demonstrated that the mutant has enhanced resistance to this pathogen compared with wild type plants. Taken together our results suggest that crk10-A397T is a gain-of-function allele of CRK10, the first such mutant to have been identified for a CRK in Arabidopsis.

Defects in meristem maintenance, cell division, and cytokinin signaling are early responses in the boron deficient maize mutant tassel-less1.

Meristems house the stem cells needed for the developmental plasticity observed in adverse environmental conditions and are crucial for determining plant architecture. Meristem development is particularly sensitive to deficiencies of the micronutrient boron, yet how boron integrates into meristem development pathways is unknown. We addressed this question using the boron-deficient maize mutant, tassel-less1 (tls1). Reduced boron uptake in tls1 leads to a progressive impairment of meristem development that manifests in vegetative and reproductive defects. We show, that the tls1 tassel phenotype (male reproductive structure) was partially suppressed by mutations in the CLAVATA1 (CLV1)-ortholog, thick tassel dwarf1 (td1), but not by other mutants in the well characterized CLV-WUSCHEL pathway, which controls meristem size. The suppression of tls1 by td1 correlates with altered signaling of the phytohormone cytokinin. In contrast, mutations in the meristem maintenance gene knotted1 (kn1) enhanced both vegetative and reproductive defects in tls1. In addition, reduced transcript levels of kn1 and cell cycle genes are early defects in tls1 tassel meristems. Our results show that specific meristem maintenance and hormone pathways are affected in tls1, and suggest that reduced boron levels induced by tls1 are the underlying cause of the observed defects. We, therefore, provide new insights into the molecular mechanisms affected by boron deficiency in maize, leading to a better understanding of how genetic and environmental factors integrate during shoot meristem development.

Carbon-11 Radiotracing Reveals Physiological and Metabolic Responses of Maize Grown under Different Regimes of Boron Treatment.

In agriculture, boron is known to play a critical role in healthy plant growth. To dissect the role of boron in maize metabolism, radioactive carbon-11 (t½ 20.4 min) was used to examine the physiological and metabolic responses of 3-week-old B73 maize plants to different levels of boron spanning 0 mM, 0.05 mM, and 0.5 mM boric acid (BA) treatments. Growth behavior, of both shoots and roots, was recorded and correlated to plant physiological responses. 11CO2 fixation, leaf export of [11C]-photosynthates, and their rate of transport increased systematically with increasing BA concentrations, while the fraction of [11C]-photosynthates delivered to the roots under 0 mM and 0.5 mM BA treatments was lower than under 0.05 mM BA treatment, likely due to changes in root growth. Additionally, solid-phase extraction coupled with gamma counting, radio-fluorescence thin layer chromatography, and radio-fluorescence high-performance liquid chromatography techniques applied to tissue extracts provided insight into the effects of BA treatment on 'new' carbon (as 11C) metabolism. Most notable was the strong influence reducing boron levels had on raising 11C partitioning into glutamine, aspartic acid, and asparagine. Altogether, the growth of maize under different regimes of boron affected 11CO2 fixation, its metabolism and allocation belowground, and altered root growth. Finally, inductively coupled plasma mass spectrometry provided insight into the effects of BA treatment on plant uptake of other essential nutrients. Here, levels of boron and zinc systematically increased in foliar tissues with increasing BA concentration. However, levels of magnesium, potassium, calcium, manganese, and iron remained unaffected by treatment. The rise in foliar zinc levels with increased BA concentration may contribute to improved 11CO2 fixation under these conditions.

Assessment of a 18F-Phenylboronic Acid Radiotracer for Imaging Boron in Maize.

Boron (B) is an essential plant micronutrient. Deficiencies of B have drastic consequences on plant development leading to crop yield losses and reductions in root and shoot growth. Understanding the molecular and cellular consequences of B deficiency is challenging, partly because of the limited availability of B imaging techniques. In this report we demonstrate the efficacy of using 4-fluorophenylboronic acid (FPBA) as a B imaging agent, which is a derivative of the B deficiency mimic phenylboronic acid (PBA). We show that radioactively labelled [18F]FPBA (t½=110 m) accumulates at the root tip, the root elongation zone and at lateral root initiation sites in maize roots, and also translocates to the shoot where it accumulates along the leaf edges. Treatment of maize seedlings using FPBA and PBA causes a shortened primary root phenotype with absence of lateral roots in a dose-dependent manner. The primary root defects can be partially rescued by the addition of boric acid indicating that PBA can be used to induce B deficiency in maize and that radioactively labelled FPBA can be used to image sites of B demand on a tissue level.

From element to development: the power of the essential micronutrient boron to shape morphological processes in plants.

Deficiency of the essential nutrient boron (B) in the soil is one of the most widespread micronutrient deficiencies worldwide, leading to developmental defects in root and shoot tissues of plants, and severe yield reductions in many crops. Despite this agricultural importance, the underlying mechanisms of how B shapes plant developmental and morphological processes are still not unequivocally understood in detail. This review evaluates experimental approaches that address our current understanding of how B influences plant morphological processes by focusing on developmental defects observed under B deficiency. We assess what is known about mechanisms that control B homeostasis and specifically highlight: (i) limitations in the methodology that is used to induce B deficiency; (ii) differences between mutant phenotypes and normal plants grown under B deficiency; and (iii) recent research on analyzing interactions between B and phytohormones. Our analysis highlights the need for standardized methodology to evaluate the roles of B in the cell wall versus other parts of the cell.

Using Xenopus laevis Oocytes to Functionally Characterize Plant Transporters.

Functionally characterizing plant membrane transport proteins is challenging. Typically, heterologous systems are used to study them. Immature eggs (oocytes) of the South African clawed frog Xenopus laevis are considered an ideal expression system for such studies. These large oocytes have a low number of endogenous transport systems in their plasma membranes and highly express foreign mRNA; the oocyte plasma membrane is the default destination of integral membrane proteins that lack recognized organellar sorting signals. These features facilitate almost background-free characterization of putative plant membrane transporters. Here we describe how to isolate Xenopus laevis oocytes, prepare capped sense RNA (cRNA) of the maize boron importer TASSEL-LESS1 (TLS1) as an example, microinject the cRNA into the isolated oocytes, and functionally assess the boron import capabilities of TLS1 in an oocyte swelling assay. These protocols can be easily adapted to study other plant and non-plant transporters with putative import function. © 2019 by John Wiley & Sons, Inc.

The barren stalk2 Gene Is Required for Axillary Meristem Development in Maize.

The diversity of plant architecture is determined by axillary meristems (AMs). AMs are produced from small groups of stem cells in the axils of leaf primordia and generate vegetative branches and reproductive inflorescences. Previous studies identified genes critical for AM development that function in auxin biosynthesis, transport, and signaling. barren stalk1 (ba1), a basic helix-loop-helix transcription factor, acts downstream of auxin to control AM formation. Here, we report the cloning and characterization of barren stalk2 (ba2), a mutant that fails to produce ears and has fewer branches and spikelets in the tassel, indicating that ba2 functions in reproductive AM development. Furthermore, the ba2 mutation suppresses tiller growth in the teosinte branched1 mutant, indicating that ba2 also plays an essential role in vegetative AM development. The ba2 gene encodes a protein that co-localizes and heterodimerizes with BA1 in the nucleus. Characterization of the genetic interaction between ba2 and ba1 demonstrates that ba1 shows a gene dosage effect in ba2 mutants, providing further evidence that BA1 and BA2 act together in the same pathway. Characterization of the molecular and genetic interaction between ba2 and additional genes required for the regulation of ba1 further supports this finding. The ba1 and ba2 genes are orthologs of rice genes, LAX PANICLE1 (LAX1) and LAX2, respectively, hence providing insights into pathways controlling AMs development in grasses.

Auxin EvoDevo: Conservation and Diversification of Genes Regulating Auxin Biosynthesis, Transport, and Signaling.

The phytohormone auxin has been shown to be of pivotal importance in growth and development of land plants. The underlying molecular players involved in auxin biosynthesis, transport, and signaling are quite well understood in Arabidopsis. However, functional characterizations of auxin-related genes in economically important crops, specifically maize and rice, are still limited. In this article, we comprehensively review recent functional studies on auxin-related genes in both maize and rice, compared with what is known in Arabidopsis, and highlight conservation and diversification of their functions. Our analysis is illustrated by phylogenetic analysis and publicly available gene expression data for each gene family, which will aid in the identification of auxin-related genes for future research. Current challenges and future directions for auxin research in maize and rice are discussed. Developments in gene editing techniques provide powerful tools for overcoming the issue of redundancy in these gene families and will undoubtedly advance auxin research in crops.

Increased transpiration is correlated with reduced boron deficiency symptoms in the maize tassel-less1 mutant.

Loss-of-function mutations of the tassel-less1 (tls1) gene in maize, which is the co-ortholog of the Arabidopsis boron (B) importer NIP5;1, leads to the loss of reproductive structures (tassels and ears). The tls1 phenotypes can be rescued by B supplementation in the field and in the greenhouse. As the rescue with B supplementation is variable in the field, we investigated additional abiotic factors, potentially causing this variation in controlled greenhouse conditions. We found that the B-dependent rescue of the tls1 mutant tassel phenotype was enhanced when plants were grown with a mix of high pressure sodium (HPS) and metal halide (MH) lamps. Normal and tls1 plants had a significant increase in transpiration and increased B content in the leaves in the greenhouse with the addition of MH lamps. Our findings imply that B transport to the shoot is enhanced through increased transpiration, which suggests that the xylem transpiration stream provides a significant supply of B in maize.

Boronic acids as tools to study (plant) developmental processes?

Boron (B) is an essential micronutrient for organisms. In plants, B is known to stabilize the cell wall by crosslinking Rhamnogalacturonan II through ester bonds formed with cis-diols of sugar moieties. However, B is believed to be required for additional functions such as stability and function of (plasma membrane) proteins involved in signal transduction pathways. We have recently shown that boronic acids, competitors of B, efficiently induce perfect phenocopies of monopteros mutants. This effect is enigmatic because like B, boronic acids should find numerous cellular targets and thus disturb many biologic processes ending in a spectrum of unspecific embryo phenotypes. Based on chemical characteristics of boronic acids and their derivatives we discuss reasons that could explain this unusual specificity. The peculiarities of this class of compounds could provide new tools for studying developmental processes.

Boronic acid treatment phenocopies monopteros by affecting PIN1 membrane stability and polar auxin transport in Arabidopsis thaliana embryos.

Several observations suggest that the micronutrient boron (B) has a stabilising role in the plasma membrane (PM), supporting functions in PM-linked (hormone) signalling processes. However, this role is poorly characterised. Here we show treatment with boronic acids, specific competitors of B, phenocopies the Arabidopsis thaliana rootless pattern mutant monopteros. At least in part, this is caused by phenylboronic acid (PBA)-induced internalisation of the membrane-localised auxin efflux carrier PINFORMED1 (PIN1) in the early embryo. PIN1 internalisation interrupts the feedback signal transduction cascade involving the phytohormone auxin, PIN1 and the transcription factor gene MONOPTEROS This entails several effects, including abnormal development of vascular cell precursors, suppression of MONOPTEROS downstream targets and loss of the root auxin maximum - essential signals for root meristem development. While PIN1 is internalised, we observe a differential effect of PBA on other proteins, which are either unaffected, internalised or, as in the case of the B transporter BOR1, stabilised at the PM. These findings suggest a competition of PBA with B for plant membrane proteins and might shed light on the function of B at the PM.

Ectopic shoot meristem generation in monocotyledonous rpk1 mutants is linked to SAM loss and altered seedling morphology.

BACKGROUND: In dicot Arabidopsis thaliana embryos two cotyledons develop largely autonomously from the shoot apical meristem (SAM). Recessive mutations in the Arabidopsis receptor-like kinase RPK1 lead to monocotyledonous seedlings, with low (10 %) penetrance due to complex functional redundancy. In strong rpk1 alleles, about 10 % of these (i. e. 1 % of all homozygotes) did not develop a SAM. We wondered whether RPK1 might also control SAM gene expression and SAM generation in addition to its known stochastic impact on cell division and PINFORMED1 (PIN1) polarity in the epidermis. RESULTS: SAM-less seedlings developed a simple morphology with a straight and continuous hypocotyl-cotyledon structure lacking a recognizable epicotyl. According to rpk1's auxin-related PIN1 defect, the seedlings displayed defects in the vascular tissue. Surprisingly, SAM-less seedlings variably expressed essential SAM specific genes along the hypocotyl-cotyledon structure up into the cotyledon lamina. Few were even capable of developing an ectopic shoot meristem (eSM) on top of the cotyledon. CONCLUSIONS: The results highlight the developmental autonomy of the SAM vs. cotyledons and suggest that the primary rpk1 defect does not lie in the seedling's ability to express SAM genes or to develop a shoot meristem. Rather, rpk1's known defects in cell division and auxin homeostasis, by disturbed PIN1 polarity, impact on SAM and organ generation. In early embryo stages this failure generates a simplified monocotyledonous morphology. Once generated, this likely entails a loss of positional information that in turn affects the spatiotemporal development of the SAM. SAM-bearing and SAM-less monocotyledonous phenotypes show morphological similarities either to real monocots or to dicot species, which only develop one cotyledon. The specific cotyledon defect in rpk1 mutants thus sheds light upon the developmental implications of the transition from two cotyledons to one.

Mouse IDGenes: a reference database for genetic interactions in the developing mouse brain.

The study of developmental processes in the mouse and other vertebrates includes the understanding of patterning along the anterior-posterior, dorsal-ventral and medial- lateral axis. Specifically, neural development is also of great clinical relevance because several human neuropsychiatric disorders such as schizophrenia, autism disorders or drug addiction and also brain malformations are thought to have neurodevelopmental origins, i.e. pathogenesis initiates during childhood and adolescence. Impacts during early neurodevelopment might also predispose to late-onset neurodegenerative disorders, such as Parkinson's disease. The neural tube develops from its precursor tissue, the neural plate, in a patterning process that is determined by compartmentalization into morphogenetic units, the action of local signaling centers and a well-defined and locally restricted expression of genes and their interactions. While public databases provide gene expression data with spatio-temporal resolution, they usually neglect the genetic interactions that govern neural development. Here, we introduce Mouse IDGenes, a reference database for genetic interactions in the developing mouse brain. The database is highly curated and offers detailed information about gene expressions and the genetic interactions at the developing mid-/hindbrain boundary. To showcase the predictive power of interaction data, we infer new Wnt/ß-catenin target genes by machine learning and validate one of them experimentally. The database is updated regularly. Moreover, it can easily be extended by the research community. Mouse IDGenes will contribute as an important resource to the research on mouse brain development, not exclusively by offering data retrieval, but also by allowing data input. DATABASE URL: http://mouseidgenes.helmholtz-muenchen.de.

Delivering sustainable crop protection systems via the seed: exploiting natural constitutive and inducible defence pathways.

To reduce the need for seasonal inputs, crop protection will have to be delivered via the seed and other planting material. Plant secondary metabolism can be harnessed for this purpose by new breeding technologies, genetic modification and companion cropping, the latter already on-farm in sub-Saharan Africa. Secondary metabolites offer the prospect of pest management as robust as that provided by current pesticides, for which many lead compounds were, or are currently deployed as, natural products. Evidence of success and promise is given for pest management in industrial and developing agriculture. Additionally, opportunities for solving wider problems of sustainable crop protection, and also production, are discussed.

Mutations in the Arabidopsis RPK1 gene uncouple cotyledon anlagen and primordia by modulating epidermal cell shape and polarity.

Plant seedlings have either one or two cotyledons. The mechanisms that regulate this organ number are poorly understood. Mutations in the RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) gene of the dicot Arabidopsis have only one cotyledon, with low penetrance due to complex genetic redundancy. An analysis of patterning genes required for cotyledon initiation showed that these have normal expression patterns, defining the cotyledon anlagen, in rpk1. This was also true for key genes, which organize the shoot apical meristem (SAM). By contrast, epidermal cell shape and polarity were compromised in rpk1 embryos, as evidenced by disturbed polarity of the auxin efflux carrier PIN1. PIN1 is required for the establishment of auxin maxima, which induce and maintain organ primordia. The effects in rpk1 mutants manifest in a spatially and timely stochastic fashion probably due to redundancy of RPK1-like functions. Consistently, auxin maxima showed a stochastic distribution in rpk1 embryos, being at times entirely absent and at other times supernumerary. This variability may explain how monocotyledonous seedlings and cotyledon shape variants can developmentally arise in Arabidopsis and possibly in other plants.

The Arabidopsis F-box/Kelch-repeat protein At2g44130 is upregulated in giant cells and promotes nematode susceptibility.

We report that the F-box/Kelch-repeat protein At2g44130 is specifically induced by the root-knot nematode Meloidogyne incognita during the initial stages of the initiation and maintenance of the feeding site. In addition, we show that the expression of this gene promotes susceptibility of infection because knocking down the F-box gene (At2g44130) drastically reduces nematode attraction to and infection of roots. In contrast, F-box overexpressing (OE) lines had a hypersusceptible phenotype, with an increase of 34% in nematode attraction and 67% in nematode infection when grown in soil. This hypersusceptibility might be the result of an increased attraction of the second-stage juveniles toward root exudates of the F-box OE, which would suggest that the blend of compounds in the root exudates of the OE line was somewhat different from the ones present in the root exudates of the wild type and the F-box knockout and tilling lines. Although the function of the F-box/Kelch-repeat protein (At2g44130) is not known, we postulate that its activation by nematode effectors released during the infection process leads to the formation of SCF((At2g44130)) (Skp1-Cullin1-F-box protein) complexes, which are involved in facilitating successful infection by the nematode through targeting specific proteins for degradation.

Emerging roles in plant defense for cis-jasmone-induced cytochrome P450 CYP81D11.

cis-Jasmone is a volatile organic compound emitted constitutively by flowers or leaves of several plant species where it acts as an attractant for pollinators and as a chemical cue for host localisation (or avoidance) for insects. ( 1-3) It is also released by some plant species after feeding damage inflicted by herbivorous insects and in this case might serve as a chemical cue for parasitoids to guide them to their prey (so called "indirect defense"). ( 4,5) Moreover, we have recently shown that plants can perceive cis-jasmone and that it acts as a signaling molecule in A. thaliana, inducing a discrete and distinctive suite of genes, of which a large subset is putatively involved in metabolism and defense responses. ( 6) Cytochrome P450s feature prominently in these functional subsets and of these the highest fold change upon cis-jasmone treatment occurred with the cytochrome CYP81D11 (At3g28740). ( 6) Hence this gene was chosen for a more thorough analysis of the potential biological relevance of the cis-jasmone induced defense response. Although the precise function of CYP81D11 remains to be determined, we could previously demonstrate its involvement in the indirect defense response in Arabidopsis, as plants exposed to cis-jasmone ceased to be attractive to the aphid parasitoid Aphidius ervi when this P450 was inactivated by T-DNA insertion mutagenesis. ( 6) Here we report additional experiments which give further support to a role of CYP81D11 in the direct or indirect defense response of A. thaliana.

Impact of natural genetic variation on the transcriptome of autotetraploid Arabidopsis thaliana.

Polyploidy, the presence of more than two complete sets of chromosomes in an organism, has significantly shaped the genomes of angiosperms during evolution. Two forms of polyploidy are often considered: allopolyploidy, which originates from interspecies hybrids, and autopolyploidy, which originates from intraspecies genome duplication events. Besides affecting genome organization, polyploidy generates other genetic effects. Synthetic allopolyploid plants exhibit considerable transcriptome alterations, part of which are likely caused by the reunion of previously diverged regulatory hierarchies. In contrast, autopolyploids have relatively uniform genomes, suggesting lower alteration of gene expression. To evaluate the impact of intraspecies genome duplication on the transcriptome, we generated a series of unique Arabidopsis thaliana autotetraploids by using different ecotypes. A. thaliana autotetraploids show transcriptome alterations that strongly depend on their parental genome composition and include changed expression of both new genes and gene groups previously described from allopolyploid Arabidopsis. Alterations in gene expression are stable, nonstochastic, developmentally specific, and associated with changes in DNA methylation. We propose that Arabidopsis possesses an inherent and heritable ability to sense and respond to elevated, yet balanced chromosome numbers. The impact of natural variation on alteration of autotetraploid gene expression stresses its potential importance in the evolution and breeding of plants.

The transcriptome of cis-jasmone-induced resistance in Arabidopsis thaliana and its role in indirect defence.

cis-jasmone (CJ) is a plant-derived chemical that enhances direct and indirect plant defence against herbivorous insects. To study the signalling pathway behind this defence response, we performed microarray-based transcriptome analysis of CJ-treated Arabidopsis plants. CJ influenced a different set of genes from the structurally related oxylipin methyl jasmonate (MeJA), suggesting that CJ triggers a distinct signalling pathway. CJ is postulated to be biosynthetically derived from jasmonic acid, which can boost its own production through transcriptional up-regulation of the octadecanoid biosynthesis genes LOX2, AOS and OPR3. However, no effect on these genes was detected by treatment with CJ. Furthermore, CJ-responsive genes were not affected by mutations in COI1 or JAR1, which are critical signalling components in MeJA response pathway. Conversely, a significant proportion of CJ-inducible genes required the three transcription factors TGA2, TGA5 and TGA6, as well as the GRAS regulatory protein SCARECROW-like 14 (SCL14), indicating regulation by a different pathway from the classical MeJA response. Moreover, the biological importance was demonstrated in that mutations in TGA2, 5, 6, SCL14 and the CJ-inducible gene CYP81D11 blocked CJ-induced attraction of the aphid parasitoid Aphidius ervi, demonstrating that these components play a key role in CJ-induced indirect defence. Collectively, our results identify CJ as a member of the jasmonates that controls indirect plant defence through a distinct signalling pathway.

Natural variation in responsiveness of Arabidopsis thaliana to methyl jasmonate is developmentally regulated.

A number of Arabidopsis thaliana (L.) Heynh ecotypes were assayed for their responses to methyl jasmonate in order to determine any natural variation in response to this volatile signal. We observed that the regulation of methyl jasmonate-induced expression of the vegetative storage proteins VSP1 and VSP2 is linked to the developmental stage of the plants. In two ecotypes investigated further, Gr-3 and Col-0, it was observed that the VSP1/2 genes became non-responsive to methyl jasmonate stimulation as the plants progressed to bolt formation and flowering. However, the onset of when this transcriptional inactivation occurred differed between the two ecotypes, with Col-0 displaying still high levels of transcript at the onset of flowering whereas Gr-3 showed no induction of VSP1/2 transcription at the same developmental stage. To our knowledge, this is the first time that such a pattern of regulation has been described for a methyl jasmonate-regulated gene. Moreover, in an F(2) population of a cross between these two ecotypes, the trait for 'VSP1/2 methyl jasmonate non-responsiveness' segregated among individuals, indicating the feasibility of mapping the genetic components of this response.