Nectar biosynthesis is conserved among floral and extrafloral nectaries.

Nectar is a primary reward mediating plant-animal mutualisms to improve plant fitness and reproductive success. Four distinct trichomatic nectaries develop in cotton (Gossypium hirsutum), one floral and three extrafloral, and the nectars they secrete serve different purposes. Floral nectar attracts bees for promoting pollination, while extrafloral nectar attracts predatory insects as a means of indirect protection from herbivores. Cotton therefore provides an ideal system for contrasting mechanisms of nectar production and nectar composition between different nectary types. Here, we report the transcriptome and ultrastructure of the four cotton nectary types throughout development and compare these with the metabolomes of secreted nectars. Integration of these datasets supports specialization among nectary types to fulfill their ecological niche, while conserving parallel coordination of the merocrine-based and eccrine-based models of nectar biosynthesis. Nectary ultrastructures indicate an abundance of rough endoplasmic reticulum positioned parallel to the cell walls and a profusion of vesicles fusing to the plasma membranes, supporting the merocrine model of nectar biosynthesis. The eccrine-based model of nectar biosynthesis is supported by global transcriptomics data, which indicate a progression from starch biosynthesis to starch degradation and sucrose biosynthesis and secretion. Moreover, our nectary global transcriptomics data provide evidence for novel metabolic processes supporting de novo biosynthesis of amino acids secreted in trace quantities in nectars. Collectively, these data demonstrate the conservation of nectar-producing models among trichomatic and extrafloral nectaries.

New insights into the functions of carbon-calcium inclusions in plants.

Carbon-calcium inclusions (CCaI) either as calcium oxalate crystals (CaOx) or amorphous calcium carbonate cystoliths are spread among most photosynthetic organisms. They represent dynamic structures with a significant construction cost and their appearance during evolution indicates an ancient origin. Both types of inclusions share some similar functional characteristics providing adaptive advantages such as the regulation of Ca levels, and the release of CO2 and water molecules upon decomposition. The latter seems to be essential under drought conditions and explains the intense occurrence of these structures in plants thriving in dry climates. It seems, however, that for plants CaOx may represent a more prevalent storage system compared with CaCO3 due to the multifunctionality of oxalate. This compound participates in a number of important soil biogeochemical processes, creates endosymbiosis with beneficial bacteria and provides tolerance against a combination of abiotic (nutrient deprivation, metal toxicity) and biotic (pathogens, herbivores) stress factors. We suggest a re-evaluation of the roles of these fascinating plant structures under a new and holistic approach that could enhance our understanding of carbon sequestration at the whole plant level and provide future perspectives.

IRE1, a component of the unfolded protein response signaling pathway, protects pollen development in Arabidopsis from heat stress.

The unfolded protein response (UPR) is activated by various stresses during vegetative development in Arabidopsis, but is constitutively active in anthers of unstressed plants. To understand the role of the UPR during reproductive development, we analyzed a double mutant, ire1a ire1b. The double mutant knocks out the RNA-splicing arm of the UPR signaling pathway. It is fertile at room temperature but male sterile at modestly elevated temperature (ET). The conditional male sterility in the mutant is a sporophytic trait, and when the double mutant was grown at ET, defects appeared in the structure of the tapetum. As a result, the tapetum in the double mutant failed to properly deposit the pollen coat at ET, which made pollen grains clump and prevented their normal dispersal. IRE1 is a dual protein kinase/ribonuclease involved in the splicing of bZIP60 mRNA, and through complementation analysis of various mutant forms of IRE1b it was demonstrated that the ribonuclease activity of IRE1 was required for protecting male fertility from ET. It was also found that overexpression of SEC31A rescued the conditional male sterility in the double mutant. SEC31A is involved in trafficking from the endoplasmic reticulum to Golgi and a major target of the IRE1-mediated UPR signaling in stressed seedlings. Thus, IRE1, a major component of the UPR, plays an important role in protecting pollen development from ET.

Comparison of Susceptible and Resistant Maize Hybrids to Colonization by Clavibacter michiganensis subsp. nebraskensis.

Clavibacter michiganensis subsp. nebraskensis causes Goss's bacterial wilt and blight on maize and is managed primarily with C. michiganensis subsp. nebraskensis-resistant hybrids. To understand the mechanisms of resistance to infection by C. michiganensis subsp. nebraskensis, leaves of a susceptible and a resistant maize hybrid at the V4 to V5 developmental stage were wound inoculated with the pathogen. Blight lesion length was monitored, C. michiganensis subsp. nebraskensis colonizing ability was determined, and structural changes were observed using microscopy. Bacterial colonization preceded lesion development that occurred 4 to 5 days postinoculation in both hybrids. Lesion expansion in the susceptible hybrid was associated with a faster rate of C. michiganensis subsp. nebraskensis spread and multiplication in the tissues. In the resistant hybrid, spread and multiplication was reduced (P < 0.0001) and, at 16 days postinoculation, became imperceptible. Initially, C. michiganensis subsp. nebraskensis showed a preference for colonization of the metaxylem vessels in both hybrids. Spread from cell to cell was accomplished through disruption of cell walls, presumably from abundance of bacterial cells or enzymatic activity. Morphological responses of the resistant maize hybrid to infection by C. michiganensis subsp. nebraskensis were similar to those reported in maize inbred lines that were resistant to Stewart's wilt caused by Pantoea stewartii. Resistance to C. michiganensis subsp. nebraskensis was associated with production of a dense matrix in the xylem that deformed and restricted movement of the bacterial cells.

Determination of the Gelation Mechanism of Freeze-Thawed Hen Egg Yolk.

A study of yolks stored up to 168 d at -20 °C was conducted to determine the gelation behavior and mechanism of freeze-thawed yolk. Methods used were rheology, native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (native- and SDS-PAGE), differential scanning calorimetry (DSC), transmission electron microscopy (TEM), particle size analysis, and proton nuclear magnetic resonance ((1)H NMR) spectroscopy for matrix mobility. Results indicate that both constituents of plasma and granules contributed to gelation of yolk under freezing. PAGE analyses suggest that granular proteins participated in aggregation during freeze-thaw. Increasing gel strength and particle size and decreasing water and lipid-water mobility indicate that lipoproteins or apolipoproteins aggregated. At storage times ≥84 d, increased protein and lipid mobility, the detection of smaller particles, and secondarily increased gel strength suggest the liberation of protein or lipoprotein components from previously formed aggregates and further aggregation of these constituents. Disruption of the gelled yolk matrix observed with TEM supported that ice crystal formation was required for gelation to occur. A two-stage dynamic gelation model is thus proposed.

New and unusual forms of calcium oxalate raphide crystals in the plant kingdom.

Calcium oxalate crystals in higher plants occur in five major forms namely raphides, styloids, prisms, druses and crystal sand. The form, shape and occurrence of calcium oxalate crystals in plants are species- and tissue-specific, hence the presence or absence of a particular type of crystal can be used as a taxonomic character. So far, four different types of needle-like raphide crystals have been reported in plants. The present work describes two new and unusual forms of raphide crystals from the tubers of Dioscorea polystachya--six-sided needles with pointed ends (Type V) and four-sided needles with beveled ends (Type VI). Both of these new types of needles are distinct from the other four types by each having a surrounding membrane that envelopes a bundle of 10-20 closely packed thin crystalline sheets. The previously known four types of needles have solid or homogenous crystalline material, surrounded by a membrane or lamellate sheath called a crystal chamber. Only the Type VI crystals have beveled ends and the needles of the other five types have pointed ends.

Molecular Mapping of D1, D2 and ms5 Revealed Linkage between the Cotyledon Color Locus D2 and the Male-Sterile Locus ms5 in Soybean.

In soybean, genic male sterility can be utilized as a tool to develop hybrid seed. Several male-sterile, female-fertile mutants have been identified in soybean. The male-sterile, female-fertile ms5 mutant was selected after fast neutron irradiation. Male-sterility due to ms5 was associated with the "stay-green" cotyledon color mutation. The cotyledon color trait in soybean is controlled by two loci, D1 and D2. Association between cotyledon color and male-sterility can be instrumental in early phenotypic selection of sterility for hybrid seed production. The use of such selection methods saves time, money, and space, as fewer seeds need to be planted and screened for sterility. The objectives of this study were to compare anther development between male-fertile and male-sterile plants, to investigate the possible linkages among the Ms5, D1 and D2 loci, and to determine if any of the d1 or d2 mutations can be applied in hybrid seed production. The cytological analysis during anther development displayed optically clear, disintegrating microspores and enlarged, engorged pollen in the male-sterile, female-fertile ms5ms5 plants, a common characteristic of male-sterile mutants. The D1 locus was mapped to molecular linkage group (MLG) D1a and was flanked by Satt408 and BARCSOYSSR_01_1622. The ms5 and D2 loci were mapped to MLG B1 with a genetic distance ~12.8 cM between them. These results suggest that use of the d2 mutant in the selection of male-sterile line may attenuate the cost hybrid seed production in soybean.

A comparison of leaf crystal macropatterns in the two sister genera Piper and Peperomia (Piperaceae).

PREMISE OF THE STUDY: This is the first large-scale study comparing leaf crystal macropatterns of the species-rich sister genera Piper and Peperomia. It focuses on identifying types of calcium oxalate crystals and their macropatterns in leaves of both genera. The Piper results are placed in a phylogenetic context to show evolutionary patterns. This information will expand knowledge about crystals and provide specific examples to help study their form and function. One example is the first-time observation of Piper crystal sand tumbling in chlorenchyma vacuoles. METHODS: Herbarium and fresh leaves were cleared of cytoplasmic content and examined with polarizing microscopy to identify types of crystals and their macropatterns. Selected hydrated herbarium and fresh leaf punches were processed for scanning electron microscopy and x-ray elemental analysis. Vibratome sections of living Piper and Peperomia leaves were observed for anatomical features and crystal movement. KEY RESULTS: Both genera have different leaf anatomies. Piper displays four crystal types in chlorenchyma-crystal sand, raphides, styloids, and druses, whereas Peperomia displays three types-druses, raphides, and prisms. Because of different leaf anatomies and crystal types between the genera, macropatterns are completely different. Crystal macropattern evolution in both is characterized by increasing complexity, and both may use their crystals for light gathering and reflection for efficient photosynthesis under low-intensity light environments. CONCLUSIONS: Both genera have different leaf anatomies, types of crystals and crystal macropatterns. Based on Piper crystals associated with photosynthetic tissues and low-intensity light, further study of their function and association with surrounding chloroplasts is warranted, especially active crystal movement.

Peperomia leaf cell wall interface between the multiple hypodermis and crystal-containing photosynthetic layer displays unusual pit fields.

BACKGROUND AND AIMS: Leaves of succulent Peperomia obtusifolia (Piperaceae), and its related species, contain a large multilayered hypodermis (epidermis) subtended by a very small single-layered photosynthetic palisade parenchyma, the latter containing spherical aggregates of crystals called druses. Each druse is in a central vacuole surrounded by chloroplasts. All hypodermal cell walls are thin, except for thick lowermost periclinal walls associated with the upper periclinal walls of the subtending palisade cells. These thick walls display 'quilted' impressions (mounds) formed by many subtending palisade cells. Conspicuous depressions occur in most mounds, and each depression contains what appear to be many plasmodesmata. These depressions are opposite similar regions in adjacent thin palisade periclinal walls, and they can be considered special pit fields that represent thin translucent regions ('windows' or 'skylights'). Druses in the vacuoles of palisade cells occur below these pit field regions and are surrounded by conspicuous cytoplasmic chloroplasts with massive grana oriented perpendicular to the crystals, probably providing for an efficient photosynthetic system under low-intensity light. METHODS: Leaf clearings and fractures, light microscopy and crossed polarizers, general and histochemical staining, and transmission and scanning electron microscopy were used to examine these structures. KEY RESULTS: Druses in the vacuoles of palisade cells occur below the thin pit field regions in the wall interface, suggesting an interesting physical relationship that could provide a pathway for light waves, filtered through the multiple hypodermis. The light waves pass into the palisade cells and are collected and dispersed by the druses to surrounding chloroplasts with large grana. CONCLUSIONS: These results imply an intriguing possible efficient photosynthetic adaptation for species growing in low-light environments, and provide an opportunity for future research on how evolution through environmental adaptation aids plants containing crystals associated with photosynthetic tissues to exist under low-light intensity and with other stresses.

Unique calcium oxalate "duplex" and "concretion" idioblasts in leaves of tribe Naucleeae (Rubiaceae).

PREMISE OF THE STUDY: This basic study may help plant biologists better understand the variety and occurrence of crystal forms and their function in plants. Literature records hold four examples of two different crystal types in one cell. One, unillustrated report mentions druses and crystal sand from one species of tribe Naucleeae (Rubiaceae) and "occasional occurrences" in additional unnamed taxa. Here, we surveyed Naucleeae (103 of 179 species, 23 of 24 genera, all seven subtribes) for "duplex idioblast" distribution for systematic significance and describe examples of this rare feature. METHODS: Cleared, dehydrated, herbarium leaves were mounted unstained in resin. Slides were examined with polarization optics for crystal types and locations, and representative areas were electronically digitized. Scanning electron microscopy and x-ray analysis verified calcium oxalate composition. KEY RESULTS: Idioblast configurations occur as crystal sand (CS) only (most common, 92 spp.) to CS plus one embedded druse (55 spp.), to CS plus 2-3 druses (6 spp.), to one druse with scanty surrounding CS (a few spp.), to a "naked" druse (16 spp.). Trends occur in some subtribes. A previously undescribed conspicuous, spheroidal calcium oxalate aggregate "concretion" idioblast occurs in only four species of Mitragyna (Mitragynineae). Idioblasts are most common along vascular bundles and in mesophyll, less so only along vascular bundles, and least common only in mesophyll. Tiny "secondary" crystals are common in ordinary mesophyll cells. CONCLUSIONS: Crystal types appear to be systematic features in Naucleeae. Duplex idioblasts (CS and druses) and aggregate concretions are a demonstration that much is yet to be discovered about crystals.

The male sterility locus ms3 is present in a fertility controlling gene cluster in soybean.

Soybean [Glycine max (L.) Merr.] is self-pollinated. To produce large quantities of hybrid seed, insect-mediated cross-pollination is necessary. An efficient nuclear male-sterile system for hybrid seed production would benefit from molecular and/or phenotypic markers linked to male fertility/sterility loci to facilitate early identification of phenotypes. Nuclear male-sterile, female-fertile ms3 mutant is a single recessive gene and displays high outcrossed seed set with pollinators. Our objective was to map the ms3 locus. A segregating population of 150 F(2) plants from Minsoy (PI 27890) x T284H, Ms3ms3 (A00-68), was screened with 231 simple sequence repeat markers. The ms3 locus mapped to molecular linkage group (MLG) D1b (Gm02) and is flanked by markers Satt157 and Satt542, with a distance of 3.7 and 12.3 cM, respectively. Female-partial sterile-1 (Fsp1) and the Midwest Oilseed male-sterile (msMOS) mutants previously were located on MLG D1b. msMOS and Fsp1 are independent genes located very close to each other. All 3 genes are located in close proximity of Satt157. We believe that this is the first report of clustering of fertility-related genes in plants. Characterization of these closely linked genes may help in understanding the evolutionary relationship among them.

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.

Crystal macropattern development in Prunus serotina (Rosaceae, Prunoideae) leaves.

BACKGROUND AND AIMS: Prunus, subgenus Padus, exhibits two completely different calcium oxalate crystal macropatterns in mature leaves. Foliar macropattern development has been described previously in P. virginiana, representing one version. Prunus serotina, in the group exhibiting the second macropattern, is described here. The goal was to describe developmental details for comparison with P. virginiana, and to extend the sparse current knowledge of crystal macropatterns. METHODS: Leaves at various developmental stages were removed from local trees and from herbarium specimens. Early leaf stages and freehand leaf and stem sections were mounted directly in aqueous glycerine; larger leaves were processed whole or in representative pieces in household bleach, dehydrated in alcohol/xylol, and mounted in Permount. Crystals were detected microscopically between crossed polarizers. KEY RESULTS: Bud scales have a dense druse population. Druses appear first at the stipule tip and proliferate basipetally but soon stop forming; growing stipules therefore have a declining density of druses. Druses appear at the tip of leaves <1 mm long, then proliferate basipetally in the midrib. Lamina druses appear in the distal marginal teeth of leaves 3 cm long; from here they proliferate basipetally and towards midrib along major veins. In about two-thirds-grown leaves (6-9 cm length) druses are all adaxial to veins of most orders; a shift occurs then to formation of prisms, which appear first abaxial to, then all around, veins. Mature leaves have virtually all prisms encrusting all major veins, more sparsely along smaller minor veins. Late season leaves form epitactic crystals on existing prismatics. CONCLUSIONS: The developing and mature macropattern of P. serotina is almost the reverse of the pattern described previously in P. virginiana, and shows that two closely related species can develop radically different modes of crystallization. The few detailed macropattern studies to date reveal striking variations that indicate a new level of organization that must be integrated with the anatomical, physiological and molecular approaches that have been dominant so far.

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.

Oxalate and phytate concentrations in seeds of soybean cultivars [Glycine max (L.) Merr.].

This study analyzed soybean seeds from 116 cultivars for total, insoluble, and soluble oxalate (Ox), phytate (InsP6), calcium (Ca), and magnesium (Mg) because of their potential beneficial or harmful effects on human nutrition. These cultivars were divided into four groups (A-D) on the basis of the year and geographic location where they were grown. Oxalate concentration ranged from about 82 to 285 mg/100 g of dry seed. The InsP6 concentration ranged from 0.22 to 2.22 g/100 g of dry seed. There was no correlation between Ox and InsP6 within or among the four groups of cultivars. There was a significant correlation between total Ox and Ca, but not Mg, in group D cultivars (r = 0.3705; p < 0.0005). No significant relationship was found in the group A-C cultivars. Eleven group D cultivars had InsP6 less than 500 mg/100 g, but all had total Ox of 130 mg/100 g or greater. Five cultivars from groups A-C had relatively low InsP6 (group B; < or =1.01 g/100 g) and low Ox (<140 mg/100 g). These cultivars could be useful for producing soy foods beneficial to populations at risk for kidney stones and for improved mineral bioavailability. The Ox and InsP6 concentrations of the cultivars indicate that choosing specific parents could generate seeds in succeeding generations with desirable Ox and InsP6 concentrations.

Oxalate and phytate of soy foods.

The consumption of foods made from soybeans is increasing because of their desirable nutritional value. However, some soy foods contain high concentrations of oxalate and/or phytate. Oxalate is a component of calcium oxalate kidney stones, whereas phytate is an inhibitor of calcium kidney stone formation. Thirty tested commercial soy foods exhibited ranges of 0.02-2.06 mg oxalate/g and 0.80-18.79 mg phytate/g. Commercial soy foods contained 2-58 mg of total oxalate per serving and 76-528 mg phytate per serving. Eighteen of 19 tofu brands and two soymilk brands contained less than 10 mg oxalate per serving, defined as a low oxalate food. Soy flour, textured vegetable soy protein, vegetable soybeans, soy nuts, tempeh, and soynut butter exhibited greater than 10 mg per serving. The correlation between oxalate and phytate in the soy foods was significant (r = 0.71, P < 0.001) indicating that oxalate-rich soy foods also contain higher concentrations of phytate. There also was a significant correlation, based on molar basis, between the divalent ion binding potential of oxalate plus phytate and calcium plus magnesium (r = 0.90, P < 0.001) in soy foods. Soy foods containing small concentrations of oxalate and moderate concentrations of phytate may be advantageous for kidney stone patients or persons with a high risk of kidney stones.

Development of the calcium oxalate crystal macropattern in pomegranate (Punica granatum, Punicaceae).

Oxalate crystals are very common in angiosperms, but few descriptions of their macropattern (crystal types, their tissue distribution, and development) exist. Because unusually large prismatic crystals and druses, are known from pomegranate, we traced the development of crystal macropattern in various-aged leaf samples from a living plant and from herbarium specimens using unstained whole mounts (some bleached and cleared), stained leaf samples, and leaf and stem cross sections. Preparations were viewed with bright-field light microscopy and with crossed polarizers. Prismatics appear first in the subapical mid-mesophyll layer of a leaf 650 µm long. Additional prismatics form basipetally in the enlarging lamina. A preemptive wave of small prismatics appears basipetally in the midrib. Druses form secondarily acropetally in petiole and midrib, while existing lamina prismatics enlarge and new ones develop among them in mid-mesophyll. Prismatics produced early expand vertically, and many eventually extend from epidermis to epidermis. Later-formed prismatics attain intermediate sizes. No crystals form along lamina veins, but in older leaves, druses occur in spongy mesophyll, mostly near major vein junctions. In the stem, druses are restricted to phloem fibers. No phloem fibers occur in the leaf trace or midvein; therefore, petiolar and midrib druses are only in parenchyma, not in phloem.

Megagametophyte abnormalities of near-isogenic female partial-sterile soybean mutants ( Glycine max; Leguminosae).

Megagametogenesis of soybean, Glycine max (L.) Merr., cultivars Clark and Clark k2, and F1 hybrid of Clark (female parent) crossed with Clark k2 (male parent) were studied using stereo light microscopy and confocal scanning laser microscopy. Reproductive development in Clark and Clark k2 plants was compared to F1 hybrid plants. In mature pods, 6.4% of the ovules of Clark, 8.1% of the ovules of Clark k2, and 41.4% of the ovules of F1 hybrid plants were aborted. This female partial sterility was due to incomplete megagametophyte development: undeveloped polar nuclei-or developed but not in a position for fertilization; increased megagametophyte wall thickness; abnormal shape and/or premature degeneration of synergids and intact synergids throughout the life of the ovule; egg cell not well-developed or absent; and megagametophyte remaining uninucleate. Each of these abnormalities contributed to either lack of double fertilization or early megagametophyte abortion.

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 of POTH1, unlike that of most other class I knotted-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, GA(1), 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.

Microscopy of a cytoplasmic male-sterile soybean from an interspecific cross between Glycine max and G. soja (Leguminosae).

Cytoplasmic male sterility has been found independently in soybean three times since 1995, but no microscopic investigation has been published. The purpose of this microscopic study was to establish the developmental sequence leading to sterility in a cytoplasmic male-sterile soybean line that has been found to be stable under all environmental conditions tested and to demarcate the temporal and spatial parameters that result in degeneration of the microspores and pollen grains. Light microscopy showed an abnormal development and/or premature degeneration of the tapetum after meiosis II, but some pollen grains persisted until after microspore mitosis. The pollen grains never completely filled with reserves. Premature formation of the endothecium also was evident. Histochemical staining for water-insoluble carbohydrates revealed an abnormal pattern of starch deposition in anther walls that coincided with lack of pollen filling. Electron microscopy showed degeneration of the inner mitochondrial membrane in the tapetal cells as the first detectable change leading to cell degeneration. Subsequently, the tapetal endoplasmic reticulum exhibited atypical concentric rings. Pollen grains displayed mitochondria with unusually enlarged inner mitochondrial spaces, degraded plastids, a rudimentary intine, and no starch or lipid reserves. Results link mitochondrial degeneration, premature formation of the endothecium, and energy deprivation to male sterility.