Harry T. Horner

Calcium oxalate crystals in plants.

Calcium (Ca) oxalate crystals occur in many plant species and in most organs and tissues. They generally form within cells although extracellular crystals have been reported. The crystal cells or idioblasts display ultrastructural modifications which are related to crystal precipitation. Crystal formation is usually associated with membranes, chambers, or inclusions found within the cell vacuole(s). Tubules, modified plastids and enlarged nuclei also have been reported in crystal idioblasts. The Ca oxalate crystals consist of either the monohydrate whewellite form, or the dihydrate weddellite form. A number of techniques exist for the identification of calcium oxalate. X-ray diffraction, Raman microprobe analysis and infrared spectroscopy are the most accurate. Many plant crystals assumed to be Ca oxalate have never been positively identified as such. In some instances, crystals have been classified as whewellite or weddellite solely on the basis of their shape. Certain evidence indicates that crystal shape may be independent of hydration form of Ca oxalate and that the vacuole crystal chamber membranes may act to mold crystal shape; however, the actual mechanism controlling shape is unknown.Oxalic acid is formed via several major pathways. In plants, glycolate can be converted to oxalic acid. The oxidation occurs in two steps with glyoxylic acid as an intermediate and glycolic acid oxidase as the enzyme. Glyoxylic acid may be derived from enzymatic cleavage of isocitric acid. Oxaloacetate also can be split to form oxalate and acetate. Another significant precursor of oxalate in plants is L-ascorbic acid. The intermediate steps in the conversion of L-ascorbic acid to oxalate are not well defined. Oxalic acid formation in animals occurs by similar pathways and Ca oxalate crystals may be produced under certain conditions.Various functions have been attributed to plant crystal idioblasts and crystals. There is evidence that oxalate synthesis is related to ionic balance. Plant crystals thus may be a manifestation of an effort to maintain an ionic equilibrium. In many plants oxalate is metabolized very slowly or not at all and is considered to be an end product of metabolism. Plant crystal idioblasts may function as a means of removing the oxalate which may otherwise accumulate in toxic quantities. Idioblast formation is dependent on the availability of both Ca and oxalate. Under Ca stress conditions, however, crystals may be reabsorbed indicating a storage function for the idioblasts for Ca. In addition, it has been suggested that the crystals serve purely as structural supports or as a protective device against foraging animals. The purpose of this review is to present an overview of plant crystal idioblasts and Ca oxalate crystals and to include the most recent literature.ZusammenfassungCalciumoxalat-Kristalle kommen in vielen Pflanzenarten und in fast allen Teilen und Geweben vor. Im allgemeinen werden sie innerhalb der Zellen gebildet, doch sind auch extrazelluläre Kristalle beschrieben. Die Kristallzellen oder Idioblasten zeigen besondere ultrastrukturelle Spezialitäten, die mit der Kristallbildung zusammenhängen. Diese ist meist mit Membranen, Räumen oder Einschlüssen in der order den Vakuole(n) verbunden. Tubuli, modifizierte Piastiden sowie vergrößerte Zellkerne sind ebenfalls für die Idioblasten erwähnt. Die Kristalle bestehen entweder aus der Monohydrat-(Whewellit) oder der Dihydrat-form (Weddellit). Mit verschiedenen Methoden können die Calciumoxalat-Kristalle identifiziert werden, die sichersten sind Röntgenstrahlen-Diffraction, Raman Mikroprobe-Analyse und die Infrarot-Spektroskopie. Viele pflanzliche Kristalle, die als Kalciumoxalat-Kristalle angesehen werden, sind nie eindeutig bestimmt worden. In einigen Fällen wurden sie lediglich auf Grund der Form als Whewellit oder Weddellit klassifiziert; es gibt aber Hinweise, dab die Kristallform unabhängig vom Hydratationsgrad ist und daß die Membranen der Kristallkammer in der Vakuole die Form beeinflussen. Der eigentliche Kontrollmechanismus ist noch unbekannt.Oxalsäure wird auf verschiedenen Wegen in den Zellen synthetisiert, in Pflanzen kann Glycollat in Oxalsäure umgewandelt werden. Die Oxidation erfolgt in zwei Schritten mit Glyoxylsäure als Zwischenprodukt und dem Enzym Glycolsäure-Oxidase. Die Glyoxylasäure könnte durch enzymatische Spaltung der Isozitronensäure entstehen. Auch kann Oxalazetat in Oxalat und Azetat splitten. Eine weitere wichtige Ausgangssubstanz für Oxalat in Pflanzen ist die L-Ascorbin-säure, die Zwischenprodukte dieser Umbildung sind noch nicht eindeutig bekannt. Oxalsäure-Bildung erfolgt bei Tieren über ähnliche Stoffwechselwege und Calciumoxalat-Kristalle mögen in ähnlicher Weise entstehen.Verschiedene Funktionen werden den pflanzlichen Kristall-Idioblasten und Kristallen zugeschrieben. Es besteht Gewißheit, daß die Oxalat-Synthese mit der Ionen-Balance korreliert ist; so können die pflanzlichen Kristalle die Anstrengungen belegen, ein Ionen-Gleichgewicht zu erhalten. In vielen Pflanzen wird das Oxalat nur in geringem Maße oder gar nicht weiter metabolisiert, es wird als Endprodukt des Stoffwechsels angesehen. Die Kristall-Idioblasten funktionieren wohl zur Entfernung des Oxalats, das sonst toxische Konzentration erreichen würde. Die Bildung hängt von der Verfügbarkeit sowohl des Calciums wie des Oxalats ab. Unter Calcium-Mangel kann es zur Reabsorption von Kristallen kommen, ein Hinweis auf eine gewisse Speicherfunktion der Idioblasten für Ca. Zusätzlich wird vermutet, daß die Kristalle mechanische Funktion haben und ein Schutz gegen fressende Tiere darstellen. Die Absicht dieses Review ist es, eine aktuelle Übersicht über die Kristall-Idioblasten und CaOxalat-Kristalle zu geben, die neueste Literatur berücksichtigt.

Mitochondrial Aldehyde Dehydrogenase Activity Is Required for Male Fertility in Maize

Some plant cytoplasms express novel mitochondrial genes that cause male sterility. Nuclear genes that disrupt the accumulation of the corresponding mitochondrial gene products can restore fertility to such plants. The Texas (T) cytoplasm mitochondrial genome of maize expresses a novel protein, URF13, which is necessary for T cytoplasm–induced male sterility. Working in concert, functional alleles of two nuclear genes, rf1 and rf2, can restore fertility to T cytoplasm plants. Rf1 alleles, but not Rf2 alleles, reduce the accumulation of URF13. Hence, Rf2 differs from typical nuclear restorers in that it does not alter the accumulation of the mitochondrial protein necessary for T cytoplasm–induced male sterility. This study established that the rf2 gene encodes a soluble protein that accumulates in the mitochondrial matrix. Three independent lines of evidence establish that the RF2 protein is an aldehyde dehydrogenase (ALDH). The finding that T cytoplasm plants that are homozygous for the rf2-R213 allele are male sterile but accumulate normal amounts of RF2 protein that lacks normal mitochondrial (mt) ALDH activity provides strong evidence that rf2-encoded mtALDH activity is required to restore male fertility to T cytoplasm maize. Detailed genetic analyses have established that the rf2 gene also is required for anther development in normal cytoplasm maize. Hence, it appears that the rf2 gene was recruited recently to function as a nuclear restorer. ALDHs typically have very broad substrate specificities. Indeed, the RF2 protein is capable of oxidizing at least three aldehydes. Hence, the specific metabolic pathway(s) within which the rf2-encoded mtALDH acts remains to be discovered.

Overexpression of a knotted-like homeobox gene of potato alters vegetative development by decreasing gibberellin accumulation.

Potato (Solanum tuberosum) homeobox 1 (POTH1) is a class I homeobox gene isolated from an early-stage tuber cDNA library. The RNA expression pattern ofPOTH1, unlike that of most other class Iknotted-like homeobox genes, is widespread in the cells of both indeterminate and differentiated tissues. Using in situ hybridization, POTH1 transcripts were detected in meristematic cells, leaf primordia, and the vascular procambium of the young stem. Overexpression of POTH1 produced dwarf plants with altered leaf morphology. Leaves were reduced in size and displayed a “mouse-ear” phenotype. The mid-vein was less prominent, resulting in a palmate venation pattern. The overall plant height of overexpression lines was reduced due to a decrease in internode length. Levels of intermediates in the gibberellin (GA) biosynthetic pathway were altered, and the bioactive GA, GA1, was reduced by one-half in sense mutants. Accumulation of mRNA for GA 20-oxidase1, a key biosynthetic enzyme, decreased in overexpression lines. In vitro tuberization was enhanced under both short- and long-day photoperiods in several POTH1 overexpression lines. Sense lines produced more tubers at a faster rate than controls. These results imply that POTH1 mediates the development of potato by acting as a negative regulator of GA biosynthesis.

A major function of the tobacco floral nectary is defense against microbial attack

Abstract. We have characterized the major nectar protein (Nectarin I) from ornamental tobacco as a superoxide dismutase that functions to generate high levels of hydrogen peroxide in nectar. Other nectar functions include an anti-polygalacturonase activity that may be due to a polygalacturonase inhibiting protein (PGIP). We also examined the expression of defense related genes in the nectary gland by two independent methods. We isolated a sample of nectary-expressed cDNAs and found that 21% of these cDNAs were defense related clones. Finally, we examined the expression of a number of specific defense-related genes by hybridization to specific cDNAs. These results demonstrated that a number of specific defense genes were more strongly expressed in the floral nectary than in the foliage. Taken together these results indicate that the floral nectary gland can have specific functions in plant defense.

Quantitative determination of calcium oxalate and oxalate in developing seeds of soybean (Leguminosae).

Developing soybean seeds accumulate very large amounts of both soluble oxalate and insoluble crystalline calcium (Ca) oxalate. Use of two methods of detection for the determination of total, soluble, and insoluble oxalate revealed that at +16 d postfertilization, the seeds were 24% dry mass of oxalate, and three-fourths of this oxalate (18%) was bound Ca oxalate. During later seed development, the dry mass of oxalate decreased. Crystals were isolated from the seeds, and X-ray diffraction and polarizing microscopy identified them as Ca oxalate monohydrate. These crystals were a mixture of kinked and straight prismatics. Even though certain plant tissues are known to contain significant amounts of oxalate and Ca oxalate during certain periods of growth, the accumulation of oxalate during soybean seed development was surprising and raises interesting questions regarding its function.

Amyloplast to chromoplast conversion in developing ornamental tobacco floral nectaries provides sugar for nectar and antioxidants for protection

Tobacco floral nectaries undergo changes in form and function. As nectaries change from green to orange, a new pigment is expressed. Analysis demonstrated that it is β-carotene. Plastids undergo dramatic changes. Early in nectary development, they divide and by stage 9 (S9) they are engorged with starch. About S9, nectaries shift from quiescent anabolism to active catabolism resulting in starch breakdown and production of nectar sugars. Starch is replaced by osmiophilic bodies, which contain needle-like carotenoid crystals. Between S9 and S12, amyloplasts are converted to chromoplasts. Changes in carotenoids and ascorbate were assayed and are expressed at low levels early in development; however, following S9 metabolic shift, syntheses of β-carotene and ascorbate greatly increase in advance of expression of nectar redox cycle. Transcript analysis for carotenoid and ascorbate biosynthetic pathways showed that these genes are significantly expressed at S6, prior to the S9 metabolic shift. Thus, formation of antioxidants β-carotene and ascorbate after the metabolic shift is independent of transcriptional regulation. We propose that biosynthesis of these antioxidants is governed by availability of substrate molecules that arise from starch breakdown. These processes and events may be amenable to molecular manipulation to provide a better system for insect attraction, cross pollination, and hybridization.

Calcium oxalate crystal types and trends in their distribution patterns in leaves of Prunus (Rosaceae: Prunoideae)

Calcium oxalate crystal types and distribution within leaves ofPrunus sensu lato (Rosaceae; Prunoideae) were surveyed from mostly herbarium specimens (196 specimens of 131 species of all five subgenera usually recognized). Rehydrated samples were bleached, mounted unstained, and viewed microscopically between crossed polarizers. Six patterns were recognized based on crystal type and relative distribution around veins and in mesophyll. Druses predominate in four subgenera, but prismatics are most common in subgenus Padus. Prunophora and Amygdalus, considered to be the most advanced subgenera, have virtually only druses, which are almost always associated with veins. Cerasus and Laurocerasus, intermediate subgenera, have the greatest diversity of patterns, but few species with prismatics. A trend is evident from mostly mesophyll prismatics in Padus to fewer prismatics and more druses of mixed distribution in Laurocerasus and Cerasus, to mostly druses restricted to veins in Amygdalus and Prunophora.

Tobacco Nectaries Express a Novel NADPH Oxidase Implicated in the Defense of Floral Reproductive Tissues against Microorganisms

Hydrogen peroxide produced from the nectar redox cycle was shown to be a major factor contributing to inhibition of most microbial growth in floral nectar; however, this obstacle can be overcome by the floral pathogen Erwinia amylovora. To identify the source of superoxide that leads to hydrogen peroxide accumulation in nectary tissues, nectaries were stained with nitroblue tetrazolium. Superoxide production was localized near nectary pores and inhibited by diphenylene iodonium but not by cyanide or azide, suggesting that NAD(P)H oxidase is the source of superoxide. Native PAGE assays demonstrated that NADPH (not NADH) was capable of driving the production of superoxide, diphenyleneiodonium chloride was an efficient inhibitor of this activity, but cyanide and azide did not inhibit. These results confirm that the production of superoxide was due to an NADPH oxidase. The nectary enzyme complex was distinct by migration on gels from the leaf enzyme complex. Temporal expression patterns demonstrated that the superoxide production (NADPH oxidase activity) was coordinated with nectar secretion, the expression of Nectarin I (a superoxide dismutase in nectar), and the expression of NOX1, a putative gene for a nectary NADPH oxidase that was cloned from nectaries and identified as an rbohD-like NADPH oxidase. Further, in situ hybridization studies indicated that the NADPH oxidase was expressed in the early stages of flower development although superoxide was generated at later stages (after Stage 10), implicating posttranslational regulation of the NADPH oxidase in the nectary.

Ascorbic acid: a precursor of oxalate in crystal idioblasts of Yucca torreyi in liquid root culture.

Liquid‐cultured primary roots of Yucca torreyi L. (Agavaceae), which are similar to its intact roots, develop uninterrupted files of calcium oxalate crystal idioblasts with raphide bundles in their cortex, beginning just proximal to the terminal meristem. Each single file of idioblasts displays a basipetal ontogenetic sequence. [1‐14C]glycolic acid, [1‐14C]glyoxylic acid, and l‐[1‐14C]ascorbic acid, all of which are potential precursors of oxalate, were each added to different flasks that contained a sterile liquid medium and isolated roots and were allowed to interact with the roots for 45 min. After thorough washing, the roots grew for periods that extended from 1.6 h to 24 h postincorporation before being fixed for light microscope autoradiography. Autoradiography of root sections with the l‐[1‐14C]ascorbic acid at the 1.6–6.0‐h incorporation times showed concentrations of silver grains over the idioblasts, primarily over the vacuole crystal bundles and cytoplasmic plastids. The [1‐14C]glyoxalic acid– and [1‐14C]glycolic acid–labeled root sections showed a smaller amount of silver grains distributed over the entire sections, but these grains were not concentrated over the crystal idioblasts. These results strongly indicate that the l‐[1‐14C]ascorbic acid is the immediate precursor of oxalate in the crystal idioblasts of Y. torreyi primary roots and support more recent biochemical data regarding oxalate synthesis in higher plants. The use of roots in liquid culture containing uninterrupted files of developing crystal idioblasts could serve as a model system for additional biochemical, physiological, and molecular studies that seek to understand the formation and functional significance of crystal idioblasts in higher plant organs.

Oil bodies in leaf mesophyll cells of angiosperms : Overview and a selected survey

Neutral (storage) oil bodies occur in leaf mesophyll cells of many angiosperms, but their literature has been largely forgotten. We review this literature and provide a survey of 302 species and hybrids from mostly north-central US species representing 113 families. Freehand cross sections of fresh leaves stained with Sudan IV verified the presence of oil. In 71 species from 24 families we observed 1-15 oil bodies per mesophyll cell. The eudicot families Asteraceae, Caprifoliaceae, Lamiaceae, and Rosaceae had the highest number of species with oil bodies, whereas few or no species in the Apiaceae, Betulaceae, Fabaceae, and Scrophulariaceae had them. Only three of 19 monocot species sampled had oil bodies. Repeat sampling of a Malus (crabapple) cultivar and a Euonymus species showed conspicuous oil bodies in mid-summer and also in mid-autumn in both attached and recently shed leaves. Oil bodies in leaf mesophyll cells are conspicuous (visible in hand cross sections using moderate magnification in unstained water mounts) in numerous species, and they occur throughout the growing season in at least some species. Neutral oil bodies in leaf mesophyll cells are not mentioned in contemporary textbooks and advanced works, but they deserve recognition as significant cellular components of many taxa, in which they may be significant sources of commercial oils.