Genetic evidence for differences in the pathways of druse and prismatic calcium oxalate crystal formation in Medicago truncatula.

Current evidence supports a single pathway of oxalate biosynthesis utilising ascorbic acid as the precursor. In this study, we begin to address the possibility that more than one pathway of oxalate biosynthesis and calcium oxalate formation occurs in Medicago truncatula Gaertn. (cv. Jemalong genotype A17). Like the wild type, developing leaves of the calcium oxalate defective (cod) 4 mutant contain prismatic crystals along the vascular strand, but this mutant also hyper-accumulates druse crystals within the mesophyll cells. A second mutant, cod5, fails to accumulate prismatic crystals along the vascular strand, but is capable of wild type druse crystal accumulation in maturing leaves. To assess whether a single pathway of oxalate biosynthesis and calcium oxalate formation occurs in M. truncatula, we generated and characterised the cod4/cod5 double mutant. Microscopic examination of the cod4/cod5 revealed that the double mutant exhibits both cod4 and cod5 mutant crystal phenotypes simultaneously, suggesting there are differences in the pathways leading to the two crystal types. Measured ascorbic acid levels and ascorbate induction studies were consistent with the acid as precursor to oxalate in druse crystal formation but not necessarily prismatic crystal formation. On the basis of these findings, we propose a working model depicting possible pathways of oxalate biosynthesis and calcium oxalate formation.

A genetic mutation that reduces calcium oxalate content increases calcium availability in Medicago truncatula.

Oxalate is considered an antinutrient that renders calcium unavailable for nutritional absorption by humans. Efforts have been made to generate and identify edible plants with decreased levels of this antinutrient. The extent to which a food can be nutritionally improved through genetic alterations in calcium oxalate content, however, has not been determined. The recent identification of near isogenic lines of the forage legume, Medicago truncatula Gaertn. (cv. Jemalong genotype A17), that differ in calcium oxalate content aids in filling this gap in our knowledge. In this study, we use an in vitro dialysis system to show that the decrease in calcium oxalate results in an enhancement in calcium availability. By comparing virtually identical plants a more direct assignment of the calcium availability to the presence or absence of oxalate was made. In addition, this study shows, for the first time, the feasibility of improving plant foods through the genetic manipulation of its oxalate content.

Oxalate reduces calcium availability in the pads of the prickly pear cactus through formation of calcium oxalate crystals.

The pads (nopales) of the prickly pear cactus are considered to be a good source of minerals and other nutrients on the basis of compositional analysis. In this study, this analysis is taken a step further by assessing the availability of selected minerals in nopales using an in vitro digestion and dialysis method. The results obtained suggest that although nopales are enriched in a number of minerals, their tissue calcium is not freely available. Microscopic analysis, energy-dispersive X-ray microanalysis, and oxalate measurements suggest that this reduction in available calcium is a result of its sequestration in the form of calcium oxalate crystals. The issue of mineral availability in plant foods is important when the dependence of many populations around the world on plant foods as their main source of minerals and other nutrients is considered.

Calcium oxalate crystal morphology mutants from Medicago truncatula.

Plants accumulate crystals of calcium oxalate in a variety of shapes and sizes. The mechanism(s) through which a plant defines the morphology of its crystals remains unknown. To gain insight into the mechanisms regulating crystal shapes, we conducted a mutant screen to identify the genetic determinants. This is the first reported mutant screen dedicated to the identification of crystal morphology mutants. A single leaf was harvested from individual Medicago truncatula L. plants that had been chemically mutagenized. Each leaf was visually inspected, using crossed-polarized light microscopy, for alterations in crystal shape and size. Seven different crystal morphology defective ( cmd) mutants were identified. Six cmd mutants were recessive and one dominant. Genetic analysis of the six recessive mutants suggested that each mutant was affected at a different locus. Each cmd mutant represents a new locus different than any previously identified. The plant phenotype of the cmd mutants appeared similar to that of the wild type in overall growth and development. This observation, coupled with the finding that several of the mutants had drastically altered the amount of calcium they partition into the oxalate crystal, questions current hypotheses regarding crystal function. Comparisons between the mutant crystals and those present in other legumes indicated the likelihood that simple point mutations contributed to the evolution of the variations in prismatic crystal shapes commonly observed in these plants today. The availability of cmd mutants provides the opportunity to investigate aspects of crystal shape and size that have been recalcitrant to previous approaches.