Manipulation of in Vivo Sorbitol Production Alters Boron Uptake and Transport in Tobacco1

Recent evidence that some species can retranslocate boron as complexes with sugar alcohols in the phloem suggests a possible mechanism for enhancing boron efficiency. We investigated the relationship between sugar alcohol (sorbitol) content, boron uptake and distribution, and translocation of foliar-applied, isotopically enriched 10B in three lines of tobacco (Nicotiana tabacum) plants differing in sorbitol production. In tobacco line S11, transformed with sorbitol-6-phosphate dehydrogenase, the production of sorbitol was accompanied by an increase in the concentration of boron in plant tissues and an increased uptake of boron compared with either tobacco line A4, transformed with antisense orientation of sorbitol-6-phosphate dehydrogenase, or wild-type tobacco (line SR1, zero-sorbitol producer). Foliar application of 10B to mature leaves was translocated to the meristematic tissues only in line S11. These results demonstrate that the concentration of the boron-complexing sugar alcohol in the plant tissue has a significant effect on boron uptake and distribution in plants, whereas the translocation of the foliar-applied 10B from the mature leaves to the meristematic tissues verifies that boron is mobile in sorbitol-producing plants (S11) as we reported previously. This suggests that selection or transgenic generation of cultivars with an increased sugar alcohol content can result in increased boron uptake, with no apparent negative effects on short-term growth.

Transgenically Enhanced Sorbitol Synthesis Facilitates Phloem Boron Transport and Increases Tolerance of Tobacco to Boron Deficiency1

The mobility of elements within plants contributes to a plant species' tolerance of nutrient deficiencies in the soil. The genetic manipulation of within-plant nutrient movement may therefore provide a means to enhance plant growth under conditions of variable soil nutrient availability. In these experiments tobacco (Nicotiana tabacum) was engineered to synthesize sorbitol, and the resultant effect on phloem mobility of boron (B) was determined. In contrast to wild-type tobacco, transgenic tobacco plants containing sorbitol exhibit a marked increase in within-plant B mobility and a resultant increase in plant growth and yield when grown with limited or interrupted soil B supply. Growth of transgenic tobacco could be maintained by reutilization of B present in mature tissues or from B supplied as a foliar application to mature leaves. In contrast, B present in mature leaves of control tobacco lines could not be used to provide the B requirements for new plant growth. 10B-labeling experiments verified that B is phloem mobile in transgenic tobacco but is immobile in control lines. These results demonstrate that the transgenic enhancement of within-plant nutrient mobility is a viable approach to improve plant tolerance of nutrient stress.

Sorbitol-6-Phosphate Dehydrogenase Expression in Transgenic Tobacco1 : High Amounts of Sorbitol Lead to Necrotic Lesions

We analyzed transgenic tobacco (Nicotiana tabacum L.) expressing Stpd1, a cDNA encoding sorbitol-6-phosphate dehydrogenase from apple, under the control of a cauliflower mosaic virus 35S promoter. In 125 independent transformants variable amounts of sorbitol ranging from 0.2 to 130 μmol g−1 fresh weight were found. Plants that accumulated up to 2 to 3 μmol g−1 fresh weight sorbitol were phenotypically normal, with successively slower growth as sorbitol amounts increased. Plants accumulating sorbitol at 3 to 5 μmol g−1 fresh weight occasionally showed regions in which chlorophyll was partially lost, but at higher sorbitol amounts young leaves of all plants lost chlorophyll in irregular spots that developed into necrotic lesions. When sorbitol exceeded 15 to 20 μmol g−1 fresh weight, plants were infertile, and at even higher sorbitol concentrations the primary regenerants were incapable of forming roots in culture or soil. In mature plants sorbitol amounts varied with age, leaf position, and growth conditions. The appearance of lesions was correlated with high sorbitol, glucose, fructose, and starch, and low myo-inositol. Supplementing myo-inositol in seedlings and young plants prevented lesion formation. Hyperaccumulation of sorbitol, which interferes with inositol biosynthesis, seems to lead to osmotic imbalance, possibly acting as a signal affecting carbohydrate allocation and transport.

5-Lipoxygenase is phosphorylated by p38 kinase-dependent MAPKAP kinases

5-lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes, a group of inflammatory mediators derived from arachidonic acid (AA). Here we describe that activation of p38 mitogen-activated protein kinase in human polymorphonuclear leukocytes and in Mono Mac 6 cells leads to activation of downstream kinases, which can subsequently phosphorylate 5-LO in vitro. Different agents activated the 5-LO kinase activities, including stimuli for cellular leukotriene biosynthesis (A23187, thapsigargin, N-formyl-leucyl-phenylalanine), compounds that up-regulate the capacity for leukotriene biosynthesis (phorbol 12-myristate 13-acetate, tumor necrosis factor α, granulocyte/macrophage colony-stimulating factor), and well known p38 stimuli as sodium arsenite and sorbitol. For all stimuli, 5-LO kinase activation was counteracted by SB203580 (3 μM or less), an inhibitor of p38 kinase. At least two p38-dependent 5-LO kinase activities were found. Based on migration properties in in-gel kinase assays and immunoreactivity, one of these was identified as mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2). The other appeared to be MAPKAP kinase 3; however, it could not be excluded that also other p38-dependent kinases contributed. When polymorphonuclear leukocytes were incubated with sodium arsenite (strong activator of 5-LO kinases), platelet-activating factor and exogenous AA, there was a 4-fold increase in 5-LO activity as compared with incubations with only platelet-activating factor and AA. This indicates that 5-LO phosphorylation can be one factor determining cellular 5-LO activity.

Tonicity-responsive enhancer binding protein, a Rel-like protein that stimulates transcription in response to hypertonicity

Hypertonicity (most often present as high salinity) is stressful to the cells of virtually all organisms. Cells survive in a hypertonic environment by increasing the transcription of genes whose products catalyze cellular accumulation of compatible osmolytes. In mammals, the kidney medulla is normally hypertonic because of the urinary concentrating mechanism. Cellular accumulation of compatible osmolytes in the renal medulla is catalyzed by the sodium/myo-inositol cotransporter (SMIT), the sodium/chloride/betaine cotransporter, and aldose reductase (synthesis of sorbitol). The importance of compatible osmolytes is underscored by the necrotic injury of the renal medulla and subsequent renal failure that results from the inhibition of SMIT in vivo by administration of a specific inhibitor. Tonicity-responsive enhancers (TonE) play a key role in hypertonicity-induced transcriptional stimulation of SMIT, sodium/chloride/betaine cotransporter, and aldose reductase. We report the cDNA cloning of human TonE binding protein (TonEBP), a transcription factor that stimulates transcription through its binding to TonE sequences via a Rel-like DNA binding domain. Western blot and immunohistochemical analyses of cells cultured in hypertonic medium reveal that exposure to hypertonicity elicits slow activation of TonEBP, which is the result of an increase in TonEBP amount and translocation to the nucleus.

Transport of uncharged organic solutes in Xenopus oocytes expressing red cell anion exchangers (AE1s)

When expressed in Xenopus oocytes, the trout red cell anion exchanger tAE1, but not the mouse exchanger mAE1, elicited a transport of electroneutral solutes (sorbitol, urea) in addition to the expected anion exchange activity. Chimeras constructed from mAE1 and tAE1 allowed us to identify the tAE1 domains involved in the induction of these transports. Expression of tAE1 (but not mAE1) is known to generate an anion conductance associated with a taurine transport. The present data provide evidence that (i) the capacity of tAE1 and tAE1 chimeras to generate urea and sorbitol permeability also was associated with an anion conductance; (ii) the same inhibitors affected both the permeability of solutes and anion conductance; and (iii) no measurable water transport was associated with the tAE1-dependent conductance. These results support the view that fish red blood cells, to achieve cell volume regulation in response to hypotonic swelling, activate a tAE1-associated anion channel that can mediate the passive transport of taurine and electroneutral solutes.

A Selaginella lepidophylla Trehalose-6-Phosphate Synthase Complements Growth and Stress-Tolerance Defects in a Yeast tps1 Mutant1

The accumulation of the disaccharide trehalose in anhydrobiotic organisms allows them to survive severe environmental stress. A plant cDNA, SlTPS1, encoding a 109-kD protein, was isolated from the resurrection plant Selaginella lepidophylla, which accumulates high levels of trehalose. Protein-sequence comparison showed that SlTPS1 shares high similarity to trehalose-6-phosphate synthase genes from prokaryotes and eukaryotes. SlTPS1 mRNA was constitutively expressed in S. lepidophylla. DNA gel-blot analysis indicated that SlTPS1 is present as a single-copy gene. Transformation of a Saccharomyces cerevisiae tps1Δ mutant disrupted in the ScTPS1 gene with S. lepidophylla SlTPS1 restored growth on fermentable sugars and the synthesis of trehalose at high levels. Moreover, the SlTPS1 gene introduced into the tps1Δ mutant was able to complement both deficiencies: sensitivity to sublethal heat treatment at 39°C and induced thermotolerance at 50°C. The osmosensitive phenotype of the yeast tps1Δ mutant grown in NaCl and sorbitol was also restored by the SlTPS1 gene. Thus, SlTPS1 protein is a functional plant homolog capable of sustaining trehalose biosynthesis and could play a major role in stress tolerance in S. lepidophylla.

Characterization of the osmotic response element of the human aldose reductase gene promoter.

Aldose reductase (EC 1.1.1.21) catalyzes the NADPH-mediated conversion of glucose to sorbitol. The hyperglycemia of diabetes increases sorbitol production primarily through substrate availability and is thought to contribute to the pathogenesis of many diabetic complications. Increased sorbitol production can also occur at normoglycemic levels via rapid increases in aldose reductase transcription and expression, which have been shown to occur upon exposure of many cell types to hyperosmotic conditions. The induction of aldose reductase transcription and the accumulation of sorbitol, an organic osmolyte, have been shown to be part of the physiological osmoregulatory mechanism whereby renal tubular cells adjust to the intraluminal hyperosmolality during urinary concentration. Previously, to explore the mechanism regulating aldose reductase levels, we partially characterized the human aldose reductase gene promoter present in a 4.2-kb fragment upstream of the transcription initiation start site. A fragment (-192 to +31 bp) was shown to contain several elements that control the basal expression of the enzyme. In this study, we examined the entire 4.2-kb human AR gene promoter fragment by deletion mutagenesis and transfection studies for the presence of osmotic response enhancer elements. An 11-bp nucleotide sequence (TGGAAAATTAC) was located 3.7 kb upstream of the transcription initiation site that mediates hypertonicity-responsive enhancer activity. This osmotic response element (ORE) increased the expression of the chloramphenicol acetyltransferase reporter gene product 2-fold in transfected HepG2 cells exposed to hypertonic NaCl media as compared with isoosmotic media. A more distal homologous sequence is also described; however, this sequence has no osmotic enhancer activity in transfected cells. Specific ORE mutant constructs, gel shift, and DNA fragment competition studies confirm the nature of the element and identify specific nucleotides essential for enhancer activity. A plasmid construct containing three repeat OREs and a heterologous promoter increased expression 8-fold in isoosmotic media and an additional 4-fold when the transfected cells are subjected to hyperosmotic stress (total approximately 30-fold). These findings will permit future studies to identify the transcription factors involved in the normal regulatory response mechanism to hypertonicity and to identify whether and how this response is altered in a variety of pathologic states, including diabetes.

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

The progress toward single-dose vaccines has been limited by the poor solid-state stability of vaccine antigens within controlled-release polymers, such as poly(lactide-co-glycolide). For example, herein we report that lyophilized tetanus toxoid aggregates during incubation at 37 degrees C and elevated humidity--i.e., conditions relevant to its release from such systems. The mechanism and extent of this aggregation are dependent on the moisture level in the solid protein, with maximum aggregation observed at intermediate moisture contents. The main aggregation pathway is consistent with formaldehyde-mediated cross-linking, where reactive electrophiles created and stored in the vaccine upon formalinization (exposure to formaldehyde during vaccine preparation) react with nucleophiles of a second vaccine molecule to form intermolecular cross-links. This process is inhibited by the following: (i) succinylating the vaccine to block reactive amino groups; (ii) treating the vaccine with sodium cyanoborohydride, which presumably reduces Schiff bases and some other electrophiles created upon formalinization; and (iii) addition of low-molecular-weight excipients, particularly sorbitol. The moisture-induced aggregation of another formalinized vaccine, diphtheria toxoid, is also retarded by succinylation, suggesting the generality of this mechanism for formalinized vaccines. Hence, mechanistic stability studies of the type described herein may be important for the development of effective single-dose vaccines.