David P. Livingston

Fructan and its relationship to abiotic stress tolerance in plants.

Numerous studies have been published that attempted to correlate fructan concentrations with freezing and drought tolerance. Studies investigating the effect of fructan on liposomes indicated that a direct interaction between membranes and fructan was possible. This new area of research began to move fructan and its association with stress beyond mere correlation by confirming that fructan has the capacity to stabilize membranes during drying by inserting at least part of the polysaccharide into the lipid headgroup region of the membrane. This helps prevent leakage when water is removed from the system either during freezing or drought. When plants were transformed with the ability to synthesize fructan, a concomitant increase in drought and/or freezing tolerance was confirmed. These experiments indicate that besides an indirect effect of supplying tissues with hexose sugars, fructan has a direct protective effect that can be demonstrated by both model systems and genetic transformation.

Purification and Characterization of an Oat Fructan Exohydrolase That Preferentially Hydrolyzes [beta]-2,6-Fructans

Oat (Avena sativa cv Fulghum) fructan hydrolase was purified by ammonium sulfate precipitation and anion-exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme was purified to homogeneity as determined by the presence of a single band (43 kD) on a silver-stained sodium dodecyl sulfate-polyacrylamide gel. A mixture of [beta]-2,6-linked fructan (neokestin) isolated from oat was used as the substrate to purify fructan hydrolase. Neokestin and small degree of polymerization fructan isomers were used to characterize the substrate specificity of the purified enzyme. The purified fructan hydrolase catalyzed hydrolysis of the terminal [beta]-2,6 linkage of 6G,6-kestotetraose 3.5 times more rapidly than it hydrolyzed the terminal [beta]-2,6 linkage of 6G-kestotriose and approximately 10 times faster than it hydrolyzed the terminal [beta]-2,1 linkage of chicory inulin. Sucrose and 1-kestose were not substrates. The Km for neokestin ([beta]-2,6-linked fructans with a degree of polymerization of 7–14) hydrolysis was 2.8% (w/v), and the Vmax was 0.041 [mu]mol min-1 mL-1. The Km for hydrolysis of 6G,6-kestotetraose was 5.6% (w/v), and the Vmax was 0.138 [mu]mol min-1 mL-1. Catalysis was exolytic and by multiple chain attack. Hydrolysis of neokestin was maximal at pH 4.5 to 5.0.

Characterization of a fructan exohydrolase purified from barley stems that hydrolyzes multiple fructofuranosidic linkages

Abstract Barley ( Hordeum vulgare cv Morex) fructan exohydrolase (EC 3.2.1.80) was purified by precipitation with ammonium sulfate and chromatography on anion exchange and lectin affinity columns. The final enzyme preparation was homogenous as determined by the presence of a single band on silver stained SDS-PAGE and IEF gels. The purified protein had a molecular mass of 33 kDa and a pI of 7.8. Analyses of relative hydrolytic rates of various fructans were determined by measuring released fructose by pulsed electrochemical detection after separation of reactions by HPLC. The purified enzyme hydrolyzed β -2,1-linkages in 6 G , 1-kestotetraose, 1 and 6 G -kestotetraose, 1, 1-kestotetraose, and 1-kestotriose with relative rates of 100 : 96 : 85 : 88. This enzyme slowly hydrolyzed the β -2,6-linkages in 6 G -kestotriose and in 6 G , 6-kestotetraose and sucrose with relative rates of 5 : 4 : 3 compared to 6 G , 1-kestotetraose hydrolysis rates arbitrarily set at 100. The substrate attack pattern, determined by identifying products from hydrolysis of purified fructan tetrasaccharides, was of the multichain type. Sucrose was a mixed-type inhibitor of inulin hydrolysis.

Tissue-specific gene expression in maize seeds during colonization by Aspergillus flavus and Fusarium verticillioides.

Aspergillus flavus and Fusarium verticillioides are fungal pathogens that colonize maize kernels and produce the harmful mycotoxins aflatoxin and fumonisin, respectively. Management practice based on potential host resistance to reduce contamination by these mycotoxins has proven difficult, resulting in the need for a better understanding of the infection process by these fungi and the response of maize seeds to infection. In this study, we followed the colonization of seeds by histological methods and the transcriptional changes of two maize defence-related genes in specific seed tissues by RNA in situ hybridization. Maize kernels were inoculated with either A. flavus or F. verticillioides 21-22 days after pollination, and harvested at 4, 12, 24, 48, 72, 96 and 120 h post-inoculation. The fungi colonized all tissues of maize seed, but differed in their interactions with aleurone and germ tissues. RNA in situ hybridization showed the induction of the maize pathogenesis-related protein, maize seed (PRms) gene in the aleurone and scutellum on infection by either fungus. Transcripts of the maize sucrose synthase-encoding gene, shrunken-1 (Sh1), were observed in the embryo of non-infected kernels, but were induced on infection by each fungus in the aleurone and scutellum. By comparing histological and RNA in situ hybridization results from adjacent serial sections, we found that the transcripts of these two genes accumulated in tissue prior to the arrival of the advancing pathogens in the seeds. A knowledge of the patterns of colonization and tissue-specific gene expression in response to these fungi will be helpful in the development of resistance.

Histological analysis and 3D reconstruction of winter cereal crowns recovering from freezing: a unique response in oat (Avena sativa L.).

The crown is the below ground portion of the stem of a grass which contains meristematic cells that give rise to new shoots and roots following winter. To better understand mechanisms of survival from freezing, a histological analysis was performed on rye, wheat, barley and oat plants that had been frozen, thawed and allowed to resume growth under controlled conditions. Extensive tissue disruption and abnormal cell structure was noticed in the center of the crown of all 4 species with relatively normal cells on the outside edge of the crown. A unique visual response was found in oat in the shape of a ring of cells that stained red with Safranin. A tetrazolium analysis indicated that tissues immediately inside this ring were dead and those outside were alive. Fluorescence microscopy revealed that the barrier fluoresced with excitation between 405 and 445 nm. Three dimensional reconstruction of a cross sectional series of images indicated that the red staining cells took on a somewhat spherical shape with regions of no staining where roots entered the crown. Characterizing changes in plants recovering from freezing will help determine the genetic basis for mechanisms involved in this important aspect of winter hardiness.

3D volumes constructed from pixel-based images by digitally clearing plant and animal tissue.

Construction of three‐dimensional volumes from a series of two‐dimensional images has been restricted by the limited capacity to decrease the opacity of tissue. The use of commercial software that allows colour‐keying and manipulation of two‐dimensional images in true three‐dimensional space allowed us to construct three‐dimensional volumes from pixel‐based images of stained plant and animal tissue without generating vector information. We present three‐dimensional volumes of (1) the crown of an oat plant showing internal responses to a freezing treatment, (2) a sample of a hepatocellular carcinoma from a woodchuck liver that had been heat‐treated with computer‐guided radiofrequency ablation to induce necrosis in the central portion of the tumour, and (3) several features of a sample of mouse lung. The technique is well suited to images from large sections (greater than 1 mm) generated from paraffin‐embedded tissues. It is widely applicable, having potential to recover three‐dimensional information at virtually any resolution inherent in images generated by light microscopy, computer tomography, magnetic resonance imaging or electron microscopy.

Persistent Supercooling of Reproductive Shoots Is Enabled by Structural Ice Barriers Being Active Despite an Intact Xylem Connection

Extracellular ice nucleation usually occurs at mild subzero temperatures in most plants. For persistent supercooling of certain plant parts ice barriers are necessary to prevent the entry of ice from already frozen tissues. The reproductive shoot of Calluna vulgaris is able to supercool down to below -22°C throughout all developmental stages (shoot elongation, flowering, fruiting) despite an established xylem conductivity. After localization of the persistent ice barrier between the reproductive and vegetative shoot at the base of the pedicel by infrared differential thermal analysis, the currently unknown structural features of the ice barrier tissue were anatomically analyzed on cross and longitudinal sections. The ice barrier tissue was recognized as a 250 μm long constriction zone at the base of the pedicel that lacked pith tissue and intercellular spaces. Most cell walls in this region were thickened and contained hydrophobic substances (lignin, suberin, and cutin). A few cell walls had what appeared to be thicker cellulose inclusions. In the ice barrier tissue, the area of the xylem was as much as 5.7 times smaller than in vegetative shoots and consisted of tracheids only. The mean number of conducting units in the xylem per cross section was reduced to 3.5% of that in vegetative shoots. Diameter of conducting units and tracheid length were 70% and 60% (respectively) of that in vegetative shoots. From vegetative shoots water transport into the ice barrier must pass pit membranes that are likely impermeable to ice. Pit apertures were about 1.9 μm x 0.7 μm, which was significantly smaller than in the vegetative shoot. The peculiar anatomical features of the xylem at the base of the pedicel suggest that the diameter of pores in pit membranes could be the critical constriction for ice propagation into the persistently supercooled reproductive shoots of C. vulgaris.

Histology Atlas of the Developing Mouse Hepatobiliary Hemolymphatic Vascular System with Emphasis on Embryonic Days 11.5–18.5 and Early Postnatal Development

A critical event in embryo development is the proper formation of the vascular system, of which the hepatobiliary system plays a pivotal role. This has led researchers to use transgenic mice to identify the critical steps involved in developmental disorders associated with the hepatobiliary vascular system. Vascular development is dependent upon normal vasculogenesis, angiogenesis, and the transformation of vessels into their adult counterparts. Any alteration in vascular development has the potential to cause deformities or embryonic death. Numerous publications describe specific stages of vascular development relating to various organs, but a single resource detailing the stage-by-stage development of the vasculature pertaining to the hepatobiliary system has not been available. This comprehensive histology atlas provides hematoxylin & eosin and immunohistochemical-stained sections of the developing mouse blood and lymphatic vasculature with emphasis on the hepatobiliary system between embryonic days (E) 11.5–18.5 and the early postnatal period. Additionally, this atlas includes a 3-dimensional video representation of the E18.5 mouse venous vasculature. One of the most noteworthy findings of this atlas is the identification of the portal sinus within the mouse, which has been erroneously misinterpreted as the ductus venosus in previous publications. Although the primary purpose of this atlas is to identify normal hepatobiliary vascular development, potential embryonic abnormalities are also described.

Complex bud architecture and cell-specific chemical patterns enable supercooling of Picea abies bud primordia

Abstract Bud primordia of Picea abies, despite a frozen shoot, stay ice free down to −50 °C by a mechanism termed supercooling whose biophysical and biochemical requirements are poorly understood. Bud architecture was assessed by 3D—reconstruction, supercooling and freezing patterns by infrared video thermography, freeze dehydration and extraorgan freezing by water potential measurements, and cell‐specific chemical patterns by Raman microscopy and mass spectrometry imaging. A bowl‐like ice barrier tissue insulates primordia from entrance by intrinsic ice. Water repellent and densely packed bud scales prevent extrinsic ice penetration. At −18 °C, break‐down of supercooling was triggered by intrinsic ice nucleators whereas the ice barrier remained active. Temperature‐dependent freeze dehydration (−0.1 MPa K−1) caused accumulation of extraorgan ice masses that by rupture of the shoot, pith tissue are accommodated in large voids. The barrier tissue has exceptionally pectin‐rich cell walls and intercellular spaces, and the cell lumina were lined or filled with proteins, especially near the primordium. Primordial cells close to the barrier accumulate di, tri and tetrasaccharides. Bud architecture efficiently prevents ice penetration, but ice nucleators become active inside the primordium below a temperature threshold. Biochemical patterns indicate a complex cellular interplay enabling supercooling and the necessity for cell‐specific biochemical analysis.

Metabolic changes in Avena sativa crowns recovering from freezing.

Extensive research has been conducted on cold acclimation and freezing tolerance of fall-sown cereal plants due to their economic importance; however, little has been reported on the biochemical changes occurring over time after the freezing conditions are replaced by conditions favorable for recovery and growth such as would occur during spring. In this study, GC-MS was used to detect metabolic changes in the overwintering crown tissue of oat (Avena sativa L.) during a fourteen day time-course after freezing. Metabolomic analysis revealed increases in most amino acids, particularly proline, 5-oxoproline and arginine, which increased greatly in crowns that were frozen compared to controls and correlated very significantly with days after freezing. In contrast, sugar and sugar related metabolites were little changed by freezing, except sucrose and fructose which decreased dramatically. In frozen tissue all TCA cycle metabolites, especially citrate and malate, decreased in relation to unfrozen tissue. Alterations in some amino acid pools after freezing were similar to those observed in cold acclimation whereas most changes in sugar pools after freezing were not. These similarities and differences suggest that there are common as well as unique genetic mechanisms between these two environmental conditions that are crucial to the winter survival of plants.