Johnston MG, Lenglet A, Farmer EE
Plants are shaped by their interaction with animals, investing heavily in defences when under attack. This leads to many changes, including the production of noxious compounds, signalling to predators of the feeding herbivore, and increased defence structures. Some of these changes are not yet clearly understood. To study these effects a screen was carried out for plants with increased oxylipin levels, a precursor to the wound hormone: jasmonate. This screen found fou2 a gain-offunction mutation in the Two Pore Channel 1 (TPC1) channel. In addition to increased oxylipin levels, the plant displays a severe wound mimic phenotype at 4 weeks old, including: shortened petioles, anthocyanin accumulation and epinastic leaves; as well as a potassium starvation phenotype. Recently, it was found that fou2 also had dramatically thickened cell walls in the vasculature, especially a large increase of esterified pectin. The TPC1 channel, well-studied in animals, is a vacuolar cation channel controlled by both cytosolic and vacuolar calcium levels. The fou2 point mutation, D454N, is in the luminal calcium sensor of the channel and renders it less sensitive to calcium increase in the vacuole. This led to the reverse genetics approach of crossing fou2 with cax1-3, a mutant for the main vacuolar calcium exchanger, so as to prevent the assumed calcium accumulation in the vacuole due to fou2. Indeed, the cax1-3/fou2 double mutant appears almost wild-type. Another method of reverting the fou2 phenotype is to cross fou2 into a line which cannot produce jasmonate: aos. The aos/fou2 double mutant also reverts a majority of the fou2 phenotype.
The main aim of the project was to quantify the differences in vascular morphology between wildtype plants and fou2, and secondarily to test if the defence phenotype was also reverted in the double mutants. To accomplish this we harvested petioles from 5 week old plants, grown in short day conditions (10 h light), and embedded them in resin. The samples were sectioned at 3 µm and stained for general acidic amino acids for the cell wall (2 % toluidine blue) and for esterified pectin (0.02 % ruthenium red) for three minutes. The sections were imaged at X400 for subsequent analysis. An automated macro in ImageJ was made for quantification of cell area, cell density and amount of stain in the phloem and xylem parenchyma.
Analysis of ten biological replicates per phenotype showed that fou2 plants had a smaller cell area on average than wild-type (62.5 µm and 73.6 µm, respectively in phloem parenchyma) and a 2-fold increase in esterified pectin (38.6 % in xylem parenchyma), yet had the same cell density. This strongly suggested that fou2 cells shrunk due to the creation of a secondary cell wall, not due to a proliferation of smaller cells, which would also have had an increased quantity of stain. Both mutant lines, aos and cax1-3, also reverted the phenotype of fou2 in the vasculature, which indicated that the thickening of cell walls was both calcium- and jasmonate-dependant. It is especially interesting that aos reverts the vasculature phenotype, as the calcium imbalance likely still exists. This can refute one hypothesis that esterified pectin was created by the plant to sequester the calcium.
To demonstrate that fou2 was a true wound mimic an experiment was carried out where wild-type plants and myc2-322B, a novel mutant which exaggerates wounding-phenotypes, were repetitively wounded. These plants were examined, and preliminary data suggests that the same effect, enlarged cell walls, occurs; however to a lesser extent. Additionally, the cells show two distinct cell wall types with differing electron density, adding weight to the idea of secondary wall creation. However, more biochemical analysis is required. These data imply that wild-type plants attempt to defend their vasculature, which is a vital structure for the plant and a rich source of nutrients for herbivores, by thickening their cell walls as observed in fou2.