Microspore embryogenesis in barley: anther pre-treatment stimulates plant defence gene expression

Microspore embryogenesis (ME) is a process in which the gametophytic pollen programme of the microspore is reorientated towards a new embryo sporophytic programme. This process requires a stress treatment, usually performed in the anther or isolated microspores for several days. Despite the universal use of stress to induce ME, very few studies have addressed the physiological processes that occur in the anther during this step. To further understand the processes triggered by stress treatment, we followed the response of anthers by measuring the expression of stress-related genes in two barley (Hordeum vulgare L.) cultivars differing in their ME response. Genes encoding enzymes involved in oxidative stress (glutathione-S-transferase, GST; oxalate oxidase, OxO), in the synthesis of jasmonic acid (13-lipoxygenase, Lox; allene oxide cyclase, AOC; allene oxide synthase, AOS) and in the phenylpropanoid pathway (phenylalanine ammonia lyase, PAL), as well as those encoding PR proteins (Barwin, chitinase 2b, Chit 2b; glucanase, Gluc; basic pathogenesis-related protein 1, PR1; pathogenesis-related protein 10, PR10) were up-regulated in whole anthers upon stress treatment, indicating that anther perceives stress and reacts by triggering general plant defence mechanisms. In particular, both OxO and Chit 2b genes are good markers of anther reactivity owing to their high level of induction during the stress treatment. The effect of copper sulphate appeared to limit the expression of defence-related genes, which may be correlated with its positive effect on the yield of microspor

Microspore embryogenesis: establishment of embryo identity and pattern in culture

The developmental plasticity of plants is beautifully illustrated by the competence of the immature male gametophyte to change its developmental fate from pollen to embryo development when exposed to stress treatments in culture. This process, referred to as microspore embryogenesis, is widely exploited in plant breeding, but also provides a unique system to understand totipotency and early cell fate decisions. We summarize the major concepts that have arisen from decades of cell and molecular studies on microspore embryogenesis and put these in the context of recent experiments, as well as results obtained from the study of pollen and zygotic embryo development

A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies

Since its introduction in plants 10 years ago, the bimolecular fluorescence complementation (BiFC) method, or split-YFP (yellow fluorescent protein), has gained popularity within the plant biology field as a method to study protein-protein interactions. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. The major drawback of BiFC is that the fluorescent protein halves are prone to self-assembly independent of a protein-protein interaction event. To circumvent this problem, several modifications of the technique have been suggested, but these modifications have not lead to improvements in plant BiFC protocols. Therefore, it remains crucial to include appropriate internal controls. Our literature survey of recent BiFC studies in plants shows that most studies use inappropriate controls, and a qualitative rather than quantitative read-out of fluorescence. Therefore, we provide a cautionary note and beginner’s guideline for the setup of BiFC experiments, discussing each step of the protocol, including vector choice, plant expression systems, negative controls, and signal detection. In addition, we present our experience with BiFC with respect to self-assembly, peptide linkers, and incubation temperature. With this note, we aim to provide a guideline that will improve the quality of plant BiFC experiments

BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways

Ectopic expression of the Brassica napus BABY BOOM (BBM) AP2/ERF transcription factor is sufficient to induce spontaneous cell proliferation leading primarily to somatic embryogenesis, but also to organogenesis and callus formation. We used DNA microarray analysis in combination with a post-translationally regulated BBM:GR protein and cycloheximide to identify target genes that are directly activated by BBM expression in Arabidopsis seedlings. We show that BBM activated the expression of a largely uncharacterized set of genes encoding proteins with potential roles in transcription, cellular signaling, cell wall biosynthesis and targeted protein turnover. A number of the target genes have been shown to be expressed in meristems or to be involved in cell wall modifications associated with dividing/growing cells. One of the BBM target genes encodes an ADF/cofilin protein, ACTIN DEPOLYMERIZING FACTOR9 (ADF9). The consequences of BBM:GR activation on the actin cytoskeleton were followed using the GFP:FIMBRIN ACTIN BINDING DOMAIN2 (GFP:FABD) actin marker. Dexamethasone-mediated BBM:GR activation induced dramatic changes in actin organization resulting in the formation of dense actin networks with high turnover rates, a phenotype that is consistent with cells that are rapidly undergoing cytoplasmic reorganization. Together the data suggest that the BBM transcription factor activates a complex network of developmental pathways associated with cell proliferation and growth

Symplasmic isolation marks cell fate changes during somatic embryogenesis

Cell-to-cell signalling is a major mechanism controlling plant morphogenesis. Transport of signalling molecules through plasmodesmata is one way in which plants promote or restrict intercellular signalling over short distances. Plasmodesmata are membrane-lined pores between cells that regulate the intercellular flow of signalling molecules through changes in their size, creating symplasmic fields of connected cells. Here we examine the role of plasmodesmata and symplasmic communication in the establishment of plant cell totipotency, using somatic embryo induction from Arabidopsis explants as a model system. Cell-to-cell communication was evaluated using fluorescent tracers, supplemented with histological and ultrastructural analysis, and correlated with expression of a WOX2 embryo reporter. We showed that embryogenic cells are isolated symplasmically from non-embryogenic cells regardless of the explant type (immature zygotic embryos or seedlings) and inducer system (2,4-dichlorophenoxyacetic acid or the BABY BOOM (BBM) transcription factor), but that the symplasmic domains in different explants differ with respect to the maximum size of molecule capable of moving through the plasmodesmata. Callose deposition in plasmodesmata preceded WOX2 expression in future sites of somatic embryo development, but later was greatly reduced in WOX2-expressing domains. Callose deposition was also associated with a decrease DR5 auxin response in embryogenic tissue. Treatment of explants with the callose biosynthesis inhibitor 2-deoxy-D-glucose supressed somatic embryo formation in all three systems studied, and also blocked the observed decrease in DR5 expression. Together these data suggest that callose deposition at plasmodesmata is required for symplasmic isolation and establishment of cell totipotency in Arabidopsis

Regulatory Network of Secondary Metabolism in Brassica rapa: Insight into the Glucosinolate Pathway

Brassica rapa studies towards metabolic variation have largely been focused on the profiling of the diversity of metabolic compounds in specific crop types or regional varieties, but none aimed to identify genes with regulatory function in metabolite composition. Here we followed a genetical genomics approach to identify regulatory genes for six biosynthetic pathways of health-related phytochemicals, i.e carotenoids, tocopherols, folates, glucosinolates, flavonoids and phenylpropanoids. Leaves from six weeks-old plants of a Brassica rapa doubled haploid population, consisting of 92 genotypes, were profiled for their secondary metabolite composition, using both targeted and LC-MS-based untargeted metabolomics approaches. Furthermore, the same population was profiled for transcript variation using a microarray containing EST sequences mainly derived from three Brassica species: B. napus, B. rapa and B. oleracea. The biochemical pathway analysis was based on the network analyses of both metabolite QTLs (mQTLs) and transcript QTLs (eQTLs). Colocalization of mQTLs and eQTLs lead to the identification of candidate regulatory genes involved in the biosynthesis of carotenoids, tocopherols and glucosinolates. We subsequently focused on the well-characterized glucosinolate pathway and revealed two hotspots of co-localization of eQTLs with mQTLs in linkage groups A03 and A09. Our results indicate that such a large-scale genetical genomics approach combining transcriptomics and metabolomics data can provide new insights into the genetic regulation of metabolite composition of Brassica vegetables

Benzenesulfonamide compounds for somatic embryogenesis in plants

Benzenesulfonamide compounds potentiate 2,4-D induced embryogenesis in plants. In particular, 4-chloro-N-methyl-N-(2-methylphenyl) benzenesulfonamide and analogs induce somatic embryogenesis in plants. Methods of inducing somatic embryogenesis comprise exposing selected plant tissues, e.g. seed embryos, to auxins, e.g. 2.4-D and the benzenesulfonamide compounds. Compounds can be prepared by reacting sulfonyl chloride, an amine and pyridine in CH2CI2. Crude product is suspended in ethyl acetate and washed in sodium and potassium hydrogen sulphates and brine, then dried and filtered

Substituted dihydropyrtoines for somatic embryogenesis in plants

Cyclopentyl 2,7,7-trimethyl-5-oxo-4-(4-pyridinyl)-1,4,5,6,7,8-hexahydro-3- quinolinecarboxylate and similar compounds are potentiators of auxin-induced somatic embryogenesis in plants. In particular, the inventors have discovered certain of these compounds induce somatic embryogenesis in Arabidopsis in the presence of 2,4-D. Also tested is BAY K 8644. Methods of inducing somatic embryogenesis comprise exposing selected plant tissues, e.g. seed embryos, to auxins, e.g. 2.4-D and the compounds

Biological containment strategies for transgenic crops

Biological containment is the prevention or reduction in the spread of transgenes by modifying plant growth or development, most commonly through modification of reproductive characteristics. This review provides a summary of the current strategies for biological containment, including the use of both naturally occurring and engineered mechanisms. A wide range of strategies and their efficacy, where possible, are discussed. While some strategies are still in the conceptual phase, others are much closer to realization and application in commercial agriculture

Biological containment strategies for transgenic crops

Biological containment is the prevention or reduction in the spread of transgenes by modifying plant growth or development, most commonly through modification of reproductive characteristics. This review provides a summary of the current strategies for biological containment, including the use of both naturally occurring and engineered mechanisms. A wide range of strategies and their efficacy, where possible, are discussed. While some strategies are still in the conceptual phase, others are much closer to realization and application in commercial agriculture