Michele M. McConn

The Critical Requirement for Linolenic Acid Is Pollen Development, Not Photosynthesis, in an Arabidopsis Mutant.

The very high proportions of trienoic fatty acids found in chloroplast membranes of all higher plants suggest that these lipid structures might be essential for photosynthesis. We report here on the production of Arabidopsis triple mutants that contain negligible levels of trienoic fatty acids. Photosynthesis at 22[deg]C was barely affected, and vegetative growth of the mutants was identical with that of the wild type, demonstrating that any requirement for trienoic acyl groups in membrane structure and function is relatively subtle. Although vegetative growth and development were unaffected, the triple mutants are male sterlle and produce no seed under normal conditions. Comparisons of pollen development in wild-type and triple mutant flowers established that pollen grains in the mutant developed to the tricellular stage. Exogenous applications of [alpha]-llnolenate or jasmonate restored fertility. Taken together, the results demonstrate that the critical role of trienoic acids in the life cycle of plants is as the precursor of oxylipin, a signaling compound that regulates final maturation processes and the release of pollen.

A Mutation at the fad8 Locus of Arabidopsis Identifies a Second Chloroplast [omega]-3 Desaturase.

Two independently isolated mutations at the fad7 locus in Arabidopsis produced plants with a temperature-conditional phenotype. Leaves of fad7 mutants grown at 28[deg]C contained less than 30% of wild-type levels of trienoic fatty acids (16:3 plus 18:3) compared with more than 70% of wild-type levels for plants grown at 15[deg]C. Screening of an M2 population derived from the fad7–1 line led to the identification of a line, SH1, in which the proportion of trienoic acids was much less than in fad7 plants. The segregation pattern of F2 progeny from a cross between SH1 and wild type indicated that the additional fatty acid mutation in SH1 is at a new locus, designated fad8. In a genetic background that was wild type at the FAD7 locus, the fad8 mutation had no detectable effect on overall leaf fatty acid composition irrespective of the temperature at which plants were grown. However, fatty acid analyses of individual leaf lipids revealed small decreases in the levels of 18:3 in two chloroplast lipids. In fad8 plants grown at 22[deg]C, phospha-tidylglycerol contained 22.5% 18:3 compared with 33.5% in wild-type Arabidopsis. For sulfoquinovosyldiacylglycerol, the values were 31.4 and 44.5%, respectively. Together with information from studies of the cloned FAD8 gene (S. Gibson, V. Arondel, K. Iba, C. Somerville [1994] Plant Physiol 106: 1615–1621), these results indicate that the FAD8 locus encodes a chloroplast-localized 16:2/18:2 desaturase that has a substrate specificity similar to the FAD7 gene product but that is induced by low temperature.

Calcium oxalate crystal morphology mutants from Medicago truncatula.

Abstract. 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.

Calcium oxalate crystal formation is not essential for growth of Medicago truncatula

Abstract Plants invest a considerable amount of resources and energy into the formation of calcium oxalate crystals. A number of roles for crystal formation in plant growth and development have been assigned based on the prevalence of crystals, their spatial distribution, and the variety of crystal shapes. As a step toward determining whether crystal formation plays a critical role in plant growth and development, we characterized the growth, oxalate content, and mineral content of the c alcium o xalate d efective mutant, cod5. Examination of control plants, using light microscopy, revealed the accumulation of prismatic crystals along the vascular strands in all the different plant tissues with the exception of roots, in which no crystals were observed. In contrast, no prismatic crystals were detected in any of the different tissues of the cod5 mutant. Crystals of calcium oxalate were observed in the pods of cod5, but they were of a different morphology. Measurements of the oxalate content in the different tissues confirmed the cod5 crystal phenotype by exhibiting low oxalate levels compared to those of controls. The cod5 pods did contain measurable oxalate levels, but at levels several times lower than controls. Although compromised in its ability to accumulate crystals of calcium oxalate, cod5 exhibited growth, which was similar to that of controls. Moreover, cod5 and controls contained similar amounts of calcium, sodium, and potassium. Our findings suggest that calcium oxalate crystal formation is not essential for plant growth or development in the case of Medicago truncatula.

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.

Sequential subtractive approach facilitates identification of differentially expressed genes

Abstract In order to identify the genes expressed during calcium oxalate crystal formation, we devised a sequential subtractive protocol that allows for efficient isolation of rare differentially expressed transcripts. This new protocol combines the beneficial elements of suppression subtractive hybridization (SSH), subtractive hybridization (SH), and differential screening into a single method. The protocol was designed to increase retention of rare transcripts, efficiently remove unwanted transcripts, have the capacity to analyze numerous transcripts simultaneously, and reduce the number of false positives. The potential for this method was demonstrated through the identification of several transcripts that were differentially expressed in two c alcium o xalate d efective (cod) Medicago truncatula mutants: cod 4 (which has abundant crystal accumulation) and cod 5 (which lacks crystal accumulation). Transcripts were isolated that showed higher steady-state levels in cod 4 than cod 5. The observed differences in transcript levels appeared to occur via two different mechanisms. In most cases, the higher transcript levels were a result of an increase in the amount of transcript present in cod 4. In one instance, however, the higher steady-state transcript level resulted from a decrease in the amount of transcript present in cod 5 rather than an increase in cod 4. The protocol described is well suited for the identification of rare transcripts from complex transcript pools

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.

Influence of the calcium oxalate defective 4 (cod4) mutation on the growth, oxalate content, and calcium content of Medicago truncatula

Abstract Calcium oxalate crystal formation has been well documented in plants. The pathway(s) and regulatory mechanism(s) of crystal formation and function, however, remain largely unknown. As a step toward expanding our understanding of crystal formation and function, we characterize the oxalate over-accumulating mutant calcium oxalate defective 4 ( cod4 ). Measurements of oxalate content in the different cod4 tissues showed elevated levels in leaves, stems and pods, compared to the corresponding control tissues. Root oxalate content was similar between cod4 and controls. Microscopic examination of the cod4 tissues revealed that all of the tissues with elevated oxalate content accumulated druse crystals in addition to the prismatic crystals that are present in wild type. With the exception of pods, however, the calcium content did not increase in proportion to the increase in oxalate. Thus, more of the tissue calcium was partitioned into the crystalline form in the leaves and stems from cod4 than controls. Shoot biomass and chlorophyll measurements showed that the cod4 mutation also resulted in an overall reduction in plant growth and chlorophyll content.

Membrane Lipid Structure and Plant Function: What are The Relationships?

Membranes are of central importance to all biology. Membranes not only define the limits of cells but also allow for the subcellular compartmentation of various biochemical functions. The ability to establish chemical potential gradients across membranes is essential to photosynthesis, the primary means of energy capture in the biosphere. Explaining membrane lipid diversity is a central problem of membrane biology. In plants, as well as in animals, membrane lipids are extremely heterogeneous in the molecular species present. However, our capacity to explain how the structure lipid molecules affects membrane function is very limited. In this paper we shall summarize recent information about polyunsaturated lipid synthesis and discuss two mutant lines of Arabidopsis that have expanded our understanding of the roles of lipids in plant biology.