Plants as vectors for environmental prion transmission.

Prions cause fatal neurodegenerative diseases and exhibit remarkable durability, which engenders a wide array of potential exposure scenarios. In chronic wasting disease of deer, elk, moose, and reindeer and in scrapie of sheep and goats, prions are transmitted via environmental routes and the ability of plants to accumulate and subsequently transmit prions has been hypothesized, but not previously demonstrated. Here, we establish the ability of several crop and other plant species to take up prions via their roots and translocate them to above-ground tissues from various growth media including soils. We demonstrate that plants can accumulate prions in above-ground tissues to levels sufficient to transmit disease after oral ingestion by mice. Our results suggest plants may serve as vectors for prion transmission in the environment-a finding with implications for wildlife conservation, agriculture, and public health.

Application of Cadaverine to Inhibit Biotin Biosynthesis in Plants.

Biotin is an essential vitamin in plants. However, characterization of biotin deficiency has been limited by embryo lethality in mutants, which can only be rescued by supplementation of biotin. Here, we describe a protocol to characterize biotin deficiency in Arabidopsis thaliana through application of the polyamine cadaverine. Cadaverine induces changes in primary root growth. Protein biotinylation in Arabidopsis seedlings can be quantified through an assay similar to a western blot, in which protein biotinylation is detected by a streptavidin probe. This technique provides a chemical means of inhibiting biotin synthesis, allowing for further characterization of biotin deficiency on a physiological and molecular level.

Cadaverine regulates biotin synthesis to modulate primary root growth in Arabidopsis.

Cadaverine, a polyamine, has been linked to modification of root growth architecture and response to environmental stresses in plants. However, the molecular mechanisms that govern the regulation of root growth by cadaverine are largely unexplored. Here we conducted a forward genetic screen and isolated a mutation, cadaverine hypersensitive 3 (cdh3), which resulted in increased root-growth sensitivity to cadaverine, but not other polyamines. This mutation affects the BIO3-BIO1 biotin biosynthesis gene. Exogenous supply of biotin and a pathway intermediate downstream of BIO1, 7,8-diaminopelargonic acid, suppressed this cadaverine sensitivity phenotype. An in vitro enzyme assay showed cadaverine inhibits the BIO3-BIO1 activity. Furthermore, cadaverine-treated seedlings displayed reduced biotinylation of Biotin Carboxyl Carrier Protein 1 of the acetyl-coenzyme A carboxylase complex involved in de novo fatty acid biosynthesis, resulting in decreased accumulation of triacylglycerides. Taken together, these results revealed an unexpected role of cadaverine in the regulation of biotin biosynthesis, which leads to modulation of primary root growth of plants.

Quantitative Trait Loci for Root Growth Response to Cadaverine in Arabidopsis.

Root growth architecture is a major determinant of agricultural productivity and plant fitness in natural ecosystems. Here we describe the methods used in a Quantitative Trait Loci (QTL) study that allowed the identification of ORGANIC CATION TRANSPORTER 1 (OCT1) as a determinant of root growth response to cadaverine treatment in Arabidopsis thaliana. This protocol screens natural accessions to characterize the variation in root growth response to the naturally occurring polyamine cadaverine, then uses recombination mapping to identify loci that are responsible for the variation existing between two accessions with contrasting phenotypes.

Molecular Mechanisms of Root Gravitropism.

Plant shoots typically grow against the gravity vector to access light, whereas roots grow downward into the soil to take up water and nutrients. These gravitropic responses can be altered by developmental and environmental cues. In this review, we discuss the molecular mechanisms that govern the gravitropism of angiosperm roots, where a physical separation between sites for gravity sensing and curvature response has facilitated discovery. Gravity sensing takes place in the columella cells of the root cap, where sedimentation of starch-filled plastids (amyloplasts) triggers a pathway that results in a relocalization to the lower side of the cell of PIN proteins, which facilitate efflux of the plant hormone auxin efflux. Consequently, auxin accumulates in the lower half of the root, triggering bending of the root tip at the elongation zone. We review our understanding of the molecular mechanisms that control this process in primary roots, and discuss recent insights into the regulation of oblique growth in lateral roots and its impact on root-system architecture and soil exploration.

Cadaverine's Functional Role in Plant Development and Environmental Response.

Cadaverine derives from lysine in a pathway that is distinct from that of the other well-characterized ornithine- or arginine-derived polyamines. Despite a multitude of studies in bacterial systems, cadaverine has garnered little attention in plant research. Nonetheless, many plants have been found to synthesize it. For instance, the Leguminosae have been shown to produce cadaverine and use it as a precursor in the biosynthesis of quinolizidine alkaloids, secondary metabolites that are involved in insect defense and also display therapeutic pharmacological properties. Cadaverine is also present in the environment; it can be produced by rhizosphere and phyllosphere microbes. Markedly, exogenous cadaverine application causes alterations in root-system architecture. Previous research suggests cadaverine has a role in stress response, with groups reporting an increase in content upon exposure to heat, drought, salt, and oxidative stress. However, data regarding the role of cadaverine in stress response remains conflicted, as some plant systems show enhanced tolerance to stresses in its presence, while others show increased sensitivity to the same stresses. In this review, we summarize recent findings on the role of cadaverine in plant growth, development, and stress response. We also address the possible roles rhizosphere and phyllosphere microbes may play in the delivery of exogenous cadaverine near plant organs, and discuss our current understanding of the molecular pathways that contribute to cadaverine homeostasis and response in plants.

Ice-Cap: a method for growing Arabidopsis and tomato plants in 96-well plates for high-throughput genotyping.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day (1,2). This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time. The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.