Paul A. Nakata

Advances in our understanding of calcium oxalate crystal formation and function in plants

Abstract Calcium oxalate crystal formation in plants appears to play a central role in a variety of important functions, including tissue calcium regulation, protection from herbivory, and metal detoxification. Evidence is mounting to support ascorbic acid as the primary precursor to oxalate biosynthesis. The ascorbic acid utilized in oxalate biosynthesis is synthesized directly within the calcium oxalate crystal-accumulating cell, called the crystal idioblast. Several unique features of the crystal idioblast have been proposed as factors that influence calcium oxalate formation. These features include an abundance of endoplasmic reticulum (ER), acidic proteins, cytoskeletal components, and the intravacuolar matrix. A number of mutants defective in different aspects of calcium oxalate crystal formation have been isolated. Cellular and biochemical characterizations of the various mutants have revealed mutations affecting crystal nucleation, morphology, distribution, and/or amount. Such mutants will be useful tools in continued efforts to decipher the pathways of crystal formation and function in plants.

Medicago truncatula Mutants Demonstrate the Role of Plant Calcium Oxalate Crystals as an Effective Defense against Chewing Insects

Calcium oxalate is the most abundant insoluble mineral found in plants and its crystals have been reported in more than 200 plant families. In the barrel medic Medicago truncatula Gaertn., these crystals accumulate predominantly in a sheath surrounding secondary veins of leaves. Mutants of M. truncatula with decreased levels of calcium oxalate crystals were used to assess the defensive role of this mineral against insects. Caterpillar larvae of the beet armyworm Spodoptera exigua Hübner show a clear feeding preference for tissue from calcium oxalate-defective (cod) mutant lines cod5 and cod6 in choice test comparisons with wild-type M. truncatula. Compared to their performance on mutant lines, larvae feeding on wild-type plants with abundant calcium oxalate crystals suffer significantly reduced growth and increased mortality. Induction of wound-responsive genes appears to be normal in cod5 and cod6, indicating that these lines are not deficient in induced insect defenses. Electron micrographs of insect mouthparts indicate that the prismatic crystals in M. truncatula leaves act as physical abrasives during feeding. Food utilization measurements show that, after consumption, calcium oxalate also interferes with the conversion of plant material into insect biomass during digestion. In contrast to their detrimental effects on a chewing insect, calcium oxalate crystals do not negatively affect the performance of the pea aphid Acyrthosiphon pisum Harris, a sap-feeding insect with piercing-sucking mouthparts. The results confirm a long-held hypothesis for the defensive function of these crystals and point to the potential value of genes controlling crystal formation and localization in crop plants.

Comparison of the primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits

Near-full-length cDNA clones to the small and large subunit of the heterotetrameric potato tuber ADP-glucose pyrophosphorylase have been isolated and characterized. The missing amino terminal sequence of the small subunit has also been elucidated from its corresponding genomic clone. Primary sequence comparisons revealed that each potato subunit had less identity to each other than to their homologous subunit from other plants. It also appeared that the smaller subunit is more conserved among the different plants and the larger subunit more divergent. Amino acid comparisons of both potato tuber sequences to theEscherichia coli ADP-glucose pyrophosphorylase sequence revealed conserved regions important for both catalytic and allosteric function of the bacterial enzyme.

CRISPR/Cas9-mediated genome editing and gene replacement in plants: Transitioning from lab to field.

The CRISPR/Cas9 genome engineering system has ignited and swept through the scientific community like wildfire. Owing largely to its efficiency, specificity, and flexibility, the CRISPR/Cas9 system has quickly become the preferred genome-editing tool of plant scientists. In plants, much of the early CRISPR/Cas9 work has been limited to proof of concept and functional studies in model systems. These studies, along with those in other fields of biology, have led to the development of several utilities of CRISPR/Cas9 beyond single gene editing. Such utilities include multiplexing for inducing multiple cleavage events, controlling gene expression, and site specific transgene insertion. With much of the conceptual CRISPR/Cas9 work nearly complete, plant researchers are beginning to apply this gene editing technology for crop trait improvement. Before rational strategies can be designed to implement this technology to engineer a wide array of crops there is a need to expand the availability of crop-specific vectors, genome resources, and transformation protocols. We anticipate that these challenges will be met along with the continued evolution of the CRISPR/Cas9 system particularly in the areas of manipulation of large genomic regions, transgene-free genetic modification, development of breeding resources, discovery of gene function, and improvements upon CRISPR/Cas9 components. The CRISPR/Cas9 editing system appears poised to transform crop trait improvement.

Plant calcium oxalate crystal formation, function, and its impact on human health

Crystals of calcium oxalate have been observed among members from most taxonomic groups of photosynthetic organisms ranging from the smallest algae to the largest trees. The biological roles for calcium oxalate crystal formation in plant growth and development include high-capacity calcium regulation, protection against herbivory, and tolerance to heavy metals. Using a variety of experimental approaches researchers have begun to unravel the complex mechanisms controlling formation of this biomineral. Given the important roles for calcium oxalate formation in plant survival and the antinutrient and pathological impact on human health through its presence in plant foods, researchers are avidly seeking a more comprehensive understanding of how these crystals form. Such an understanding will be useful in efforts to design strategies aimed at improving the nutritional quality and production of plant foods.

A Previously Unknown Oxalyl-CoA Synthetase Is Important for Oxalate Catabolism in Arabidopsis

This work shows that degradation of oxalate via oxalyl-CoA synthetase is required for correct seed development and for pathogen defense in Arabidopsis. It identifies AAE3 as an oxalyl-CoA synthetase, providing support for an alternative pathway of oxalate degradation first proposed 50 years ago. Oxalate is produced by several catabolic pathways in plants. The best characterized pathway for subsequent oxalate degradation is via oxalate oxidase, but some species, such as Arabidopsis thaliana, have no oxalate oxidase activity. Previously, an alternative pathway was proposed in which oxalyl-CoA synthetase (EC 6.2.1.8) catalyzes the first step, but no gene encoding this function has been found. Here, we identify ACYL-ACTIVATING ENZYME3 (AAE3; At3g48990) from Arabidopsis as a gene encoding oxalyl-CoA synthetase. Recombinant AAE3 protein has high activity against oxalate, with Km = 149.0 ± 12.7 μM and Vmax = 11.4 ± 1.0 μmol/min/mg protein, but no detectable activity against other organic acids tested. Allelic aae3 mutants lacked oxalyl-CoA synthetase activity and were unable to degrade oxalate into CO2. Seeds of mutants accumulated oxalate to levels threefold higher than the wild type, resulting in the formation of oxalate crystals. Crystal formation was associated with seed coat defects and substantially reduced germination of mutant seeds. Leaves of mutants were damaged by exogenous oxalate and more susceptible than the wild type to infection by the fungus Sclerotinia sclerotiorum, which produces oxalate as a phytotoxin to aid infection. Our results demonstrate that, in Arabidopsis, oxalyl-CoA synthetase encoded by AAE3 is required for oxalate degradation, for normal seed development, and for defense against an oxalate-producing fungal pathogen.

Differential Regulation of ADP-Glucose Pyrophosphorylase in the Sink and Source Tissues of Potato

Expression of potato (Solanum tuberosum L.) ADP-Glc pyrophosphorylase (AGP) was analyzed to assess whether the expression patterns of the individual subunit genes play a role in effectuating AGP activity and hence starch biosynthesis. Temporal analysis revealed that the coordinate expression of the large (IAGP) and small (sAGP) subunits, which collectively make up the heterotetrameric AGP holoenzyme, is primarily under transcriptional control during tuber development. In contrast, noncoordinate expression of the subunit transcripts was evident in leaves in which the relative level of the sAGP mRNA was present at severalfold excess compared to the level of IAGP mRNA. Immunoblot analysis, however, revealed that the levels of sAGP and IAGP polypeptides were present at near equimolar amounts, indicating that a posttranscriptional event co-ordinates subunit polypeptide levels. This posttranscriptional control of subunit abundance was also evident in leaves subjected to a photoperiod regime and during sucrose-induced starch synthesis. The predominant role of transcriptional and posttranscriptional regulation of AGP in tubers and leaves, respectively, is consistent with the distinct pathways of carbon partitioning and with the type and function of starch synthesis that occurs within each tissue.

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.

Calcium oxalate crystal morphology.

Plants invest considerable resources of carbon and calcium in crystal formation, indicating that it is an important basic process in growth and development. The diversity of crystal shapes, as well as their prevalence and spatial distribution, have led to several hypotheses regarding crystal function in plants. The proposed functions include roles in ion balance, in plant defense, in tissue support, in detoxification, and in light gathering and reflection [1xCalcium oxalate crystals in plants. Franceschi, V.R. and Horner, H.T. Bot. Rev. 1980; 46: 361–427Crossref | Scopus (480)See all References][1]. The primary role of crystal formation might vary depending on the plant in question. Although calcium oxalate crystal formation has intrigued scientist for many years, knowledge of many aspects of its formation and function are unknown. Crystal formation does not appear to be a simple random precipitation event [2xCell-mediated crystallization of calcium oxalate in plants. Webb, M.A. Plant Cell. 1999; 11: 751–761PubMedSee all References][2]. Instead, crystals of specific morphologies are formed in a controlled and defined fashion. Most crystals can be classified into one of five categories based on their morphology: crystal sand, raphide, druse, styloid and prismatic [1xCalcium oxalate crystals in plants. Franceschi, V.R. and Horner, H.T. Bot. Rev. 1980; 46: 361–427Crossref | Scopus (480)See all References][1]. Specific variations in crystal shape can occur within each classification. Such variations of the prismatic crystal morphology can be observed in the leaves of different legumes, such as Trifolium pratense, Vigna unguiculata and Vicia faba (Fig. 1Fig. 1). The mechanisms controlling the morphology of a crystal are unknown.Fig. 1A comparison of crystals from selected legumes. Scanning electron micrographs of calcium oxalate crystals from Trifolium pratense (a), Vigna unguiculata (b), Vicia faba (c), wild-type Medicago truncatula (d) and from M. truncatula crystal morphology defective mutatnts cmd 4 (e) and cmd 6 (f). Each of the crystal shapes from the M. truncatula plants (d–f) match those present in other legumes (a–c). Scale bar = 1 μm (a, b, d–f) and 10 μm (c).View Large Image | Download PowerPoint SlideAs a step toward elucidating the mechanisms regulating crystal morphologies, my laboratory has recently initiated a mutant screen using the model legume, Medicago truncatula. To date, the screen has led to the identification of several crystal morphology defective (cmd) mutants [3xSee all References][3]. Characterization of the cmd mutants has revealed that the introduction of a single point mutation, through EMS-mutagenesis, can lead to the generation of M. truncatula plants with crystal shapes that match those present in other legumes (Fig. 1Fig. 1). For example, the crystals present in cmd 4 and cmd 6 appear similar in overall morphology to those of Vigna unguiculata and Vicia faba, respectively (Fig. 1Fig. 1). Wild-type M. truncatula crystals look identical to Trifolium pratense (Fig. 1Fig. 1). Based on such comparisons, we hypothesize that simple point mutations contributed to the evolution of the various prismatic crystal morphologies commonly observed in legumes today. Such findings emphasize that the control of the morphology of a crystal is a tightly regulated genetic process and that a simple point mutation can drastically alter the size and shape of the crystal. The availability of the cmd mutants should open new avenues of investigation into the mechanisms regulating crystal shapes and sizes.