Isolation of Medicago truncatula mutants defective in calcium oxalate crystal formation

Abstract

Plants accumulate crystals of calcium oxalate in a variety of shapes, sizes, amounts, and spatial locations. How and why many plants form crystals of calcium oxalate remain largely unknown. To gain insight into the regulatory mechanisms of crystal formation and function, we have initiated a mutant screen to identify the genetic determinants. Leaves from a chemically mutagenized Medicago truncatula population were visually screened for alterations in calcium oxalate crystal formation. Seven different classes of calcium oxalate defective mutants were identified that exhibited alterations in crystal nucleation, morphology, distribution and/or amount. Genetic analysis suggested that crystal formation is a complex process involving more than seven loci. Phenotypic analysis of a mutant that lacks crystals, cod 5, did not reveal any difference in plant growth and development compared with controls. This finding brings into question the hypothesized roles of calcium oxalate formation in supporting tissue structure and in regulating excess tissue calcium.

Figure 1

Crystal development in leaves of M. truncatula. Leaves were harvested, cleared, and visually inspected using light microscopy. A representative region of a secondary vein and surrounding mesophyll is shown in A, B, C, and D. A single expanding leaf (cleared whole mount) is shown under bright field (A) and between crossed polarizers (B). Scale bar = 10 μm. Prismatic calcium oxalate crystal (white coffin-shaped structures) were observed accumulating exclusively along the vascular strand. A whole-mount comparison between an expanding leaf (C) and a fully expanded leaf (D) is shown between crossed polarizers. In addition to the prismatic crystals present along the vascular strand, small globular-shaped crystals (white spots) were observed sparsely distributed within the mesophyll cells of the more mature leaf (D). Scale bar = 20 μm. VS, Vascular strand; GC, guard cell; M, mesophyll; Cr, calcium oxalate crystal.

Figure 2

cod mutants. A, Control plant (C) and individual cod mutants (1–7) grown under greenhouse conditions. B, A representative portion of a whole-mount leaf clearing, showing a secondary vein and surrounding mesophyll cells, from the control plant and each cod mutant (1–7) are shown under polarizing optics. Crystals appear as bright spots. The mutants cod 1, cod 4, and cod 5 showed changes in crystal number compared with controls. Mutants cod 3, cod 4, and cod 5 showed changes in the pattern of crystal distribution. Changes in crystal size were observed in cod 1, cod 3, and cod 6, whereas changes in crystal shape were observed in cod 1, cod 2, cod 3, cod 6, and cod 7. All mutants shown were backcrossed a minimum of two times and are of the same age. VS, Vascular strand; M, mesophyll; Cr, calcium oxalate crystal. Scale bars = 10 μm.