Riet De Rycke

An Agrobacterium-mediated transient gene expression system for intact leaves

Abstract An efficient and reproducible Agrobacterium -mediated transient gene expression system for intact leaf tissue was developed. A high level of transient expression was observed when bacteria, which were pretreated in vir gene-inducing conditions, were infiltrated into complete leaf tissue. Histochemical β-glucuronidase assays showed large transgene-expressing sectors comprising of up to 90% of the leaf area. As a consequence of infiltration, the induced bacteria entered into the intercellular spaces, thus cnabling T-DNA transfer in all cell layers of the leaf. The protocol was optimized for Phascolus vulgaris leaves, but similar results were obtained with other plant species, such as Phaseolus acutifolius , poplar, and tobacco. A β-glucuronidase chimeric gene interrupted by an intron was used as a marker for histological detection of the sectors.

Neutrophil extracellular trap cell death requires both autophagy and superoxide generation

Neutrophil extracellular traps (NETs) are extracellular chromatin structures that can trap and degrade microbes. They arise from neutrophils that have activated a cell death program called NET cell death, or NETosis. Activation of NETosis has been shown to involve NADPH oxidase activity, disintegration of the nuclear envelope and most granule membranes, decondensation of nuclear chromatin and formation of NETs. We report that in phorbol myristate acetate (PMA)-stimulated neutrophils, intracellular chromatin decondensation and NET formation follow autophagy and superoxide production, both of which are required to mediate PMA-induced NETosis and occur independently of each other. Neutrophils from patients with chronic granulomatous disease, which lack NADPH oxidase activity, still exhibit PMA-induced autophagy. Conversely, PMA-induced NADPH oxidase activity is not affected by pharmacological inhibition of autophagy. Interestingly, inhibition of either autophagy or NADPH oxidase prevents intracellular chromatin decondensation, which is essential for NETosis and NET formation, and results in cell death characterized by hallmarks of apoptosis. These results indicate that apoptosis might function as a backup program for NETosis when autophagy or NADPH oxidase activity is prevented.

Molecular Phenotyping of the pal1 and pal2 Mutants of Arabidopsis thaliana Reveals Far-Reaching Consequences on Phenylpropanoid, Amino Acid, and Carbohydrate Metabolism

The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (PAL), is encoded by four genes in Arabidopsis thaliana. Whereas PAL function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis pal1 and pal2 single mutants and with limited phenotypic alterations in the pal1 pal2 double mutant, significant modifications occur in the transcriptome and metabolome of the pal mutants. The disruption of PAL led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of PAL1 and PAL2 are delineated, and PAL1 is qualified as being more important for the generation of phenylpropanoids.

Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure.

Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula × Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.

A Mutation of the Mitochondrial ABC Transporter Sta1 Leads to Dwarfism and Chlorosis in the Arabidopsis Mutant starik

A mutation in the Arabidopsis gene STARIK leads to dwarfism and chlorosis of plants with an altered morphology of leaf and cell nuclei. We show that the STARIK gene encodes the mitochondrial ABC transporter Sta1 that belongs to a subfamily of Arabidopsis half-ABC transporters. The severity of the starik phenotype is suppressed by the ectopic expression of the STA2 homolog; thus, Sta1 function is partially redundant. Sta1 supports the maturation of cytosolic Fe/S protein in Δatm1 yeast, substituting for the ABC transporter Atm1p. Similar to Atm1p-deficient yeast, mitochondria of the starik mutant accumulated more nonheme, nonprotein iron than did wild-type organelles. We further show that plant mitochondria contain a putative l-cysteine desulfurase. Taken together, our results suggest that plant mitochondria possess an evolutionarily conserved Fe/S cluster biosynthesis pathway, which is linked to the intracellular iron homeostasis by the function of Atm1p-like ABC transporters.

Developmental Stage Specificity and the Role of Mitochondrial Metabolism in the Response of Arabidopsis Leaves to Prolonged Mild Osmotic Stress

When subjected to stress, plants reprogram their growth by largely unknown mechanisms. To provide insights into this process, the growth of Arabidopsis (Arabidopsis thaliana) leaves that develop under mild osmotic stress was studied. Early during leaf development, cell number and size were reduced by stress, but growth was remarkably adaptable, as division and expansion rates were identical to controls within a few days of leaf initiation. To investigate the molecular basis of the observed adaptability, leaves with only proliferating, exclusively expanding, and mature cells were analyzed by transcriptomics and targeted metabolomics. The stress response measured in growing and mature leaves was largely distinct; several hundred transcripts and multiple metabolites responded exclusively in the proliferating and/or expanding leaves. Only a few genes were differentially expressed across the three stages. Data analysis showed that proliferation and expansion were regulated by common regulatory circuits, involving ethylene and gibberellins but not abscisic acid. The role of ethylene was supported by the analysis of ethylene-insensitive mutants. Exclusively in proliferating cells, stress induced genes of the so-called “mitochondrial dysfunction regulon,” comprising alternative oxidase. Up-regulation for eight of these genes was confirmed with promoter:β-glucuronidase reporter lines. Furthermore, mitochondria of stress-treated dividing cells were morphologically distinct from control ones, and growth of plants overexpressing the alternative oxidase gene was more tolerant to osmotic and drought stresses. Taken together, our data underline the value of analyzing stress responses in development and demonstrate the importance of mitochondrial respiration for sustaining cell proliferation under osmotic stress conditions.

PtABI3 Impinges on the Growth and Differentiation of Embryonic Leaves during Bud Set in Poplar

The Arabidopsis ABSCISIC ACID–INSENSITIVE3 (ABI3) protein plays a crucial role during late seed development and has an additional function at the vegetative meristem, particularly during periods of growth-arresting conditions and quiescence. Here, we show that the ABI3 homolog of poplar (PtABI3) is expressed in buds during natural bud set. Expression occurs clearly after perception of the critical daylength that initiates bud set and dormancy in poplar. In short-day conditions mimicking natural bud set, the expression of a chimeric PtABI3::β-glucuronidase (GUS) gene occurred in those organs and cells of the apex that grow actively but will undergo arrest: the young embryonic leaves, the subapical meristem, and the procambial strands. If PtABI3 is overexpressed or downregulated, bud development in short-day conditions is altered. Constitutive overexpression of PtABI3 resulted in apical buds with large embryonic leaves and small stipules, whereas in antisense lines, bud scales were large and leaves were small. Thus, PtABI3 influences the size and ratio of embryonic leaves and bud scales/stipules that differentiate from the primordia under short-day conditions. These observations, together with the expression of PtABI3::GUS in embryonic leaves but not in bud scales/stipules, support the idea that wild-type PtABI3 is required for the relative growth rate and differentiation of embryonic leaves inside the bud. These experiments reveal that ABI3 plays a role in the cellular differentiation of vegetative tissues, in addition to its function in seeds.

Polyelectrolyte Microcapsules as Antigen Delivery Vehicles To Dendritic Cells: Uptake, Processing, and Cross‐Presentation of Encapsulated Antigens

Degradable polyelectrolyte microcapsules (PMs; see picture) as antigen delivery vehicles are taken up by dendritic cells (DCs) by macropinocytosis. Following uptake, the shell of the microcapsules ruptures, resulting in the invasion of the capsules by the cellular cytoplasm, thus allowing DCs to efficiently process encapsulated antigen.

Reactive oxygen species and ethylene play a positive role in lateral root base nodulation of a semiaquatic legume

Lateral root base nodulation on the tropical, semiaquatic legume Sesbania rostrata results from two coordinated, Nod factor-dependent processes: formation of intercellular infection pockets and induction of cell division. Infection pocket formation is associated with cell death and production of hydrogen peroxide. Pharmacological experiments showed that ethylene and reactive oxygen species mediate Nod factor responses and are required for nodule initiation, whereby induction of division and infection could not be uncoupled. Application of purified Nod factors triggered cell division, and both Nod factors and ethylene induced cavities and cell death features in the root cortex. Thus, in S. rostrata, ethylene and reactive oxygen species act downstream from the Nod factors in pathways that lead to formation of infection pockets and initiation of nodule primordia.

Histological study of seed coat development in Arabidopsis thaliana.

Arabidopsis seed coat development using light and transmission electron microscopy revealed major morphological changes associated with the transition of the integuments into the mature seed coat. By the use of a metachromatic staining procedure, cytological events such as the production of phenolic compounds and acidic polysaccharides were followed. Immediately after fertilization, the cells of the inner epidermis of the inner integument became vacuolated and subsequently accumulated pigment within them. This pigment started to disappear from the cytoplasm at the torpedo stage of the embryo, as it became green. During the torpedo stage, mucilage began to accumulate in the cells of the external epidermis of the outer integument. Furthermore, starch grains accumulated against the central part of the inner periclinal wall of these cells, resulting in the formation of small pyramidal domes that persisted until seed maturity. At the maturation stage, when the embryo became dormant and colourless, a new pigment accumulation was observed in an amorphous layer derived from remnants of crushed integument layers. This second pigment layer was responsible for the brown seed colour. These results show that seed coat formation may proceed in a coordinated way with the developmental phases of embryogenesis.