The expression of small heat shock proteins in seeds responds to discrete developmental signals and suggests a general protective role in desiccation tolerance.

To learn more about the function and regulation of small heat shock proteins (sHSPs) during seed development, we studied sHSP expression in wild-type and seed maturation mutants of Arabidopsis by western analysis and using an HSP17.4 promoter-driven beta-glucuronidase (GUS) reporter gene in transgenic plants. In the absence of stress, GUS activity increases during development until the entire embryo is stained before desiccation. Heat-stressed embryos stained for GUS at all stages, including early stages that showed no detectable HSP17. 4::GUS activity without heat. Examination of HSP17.4 expression in seeds of the transcriptional activator mutants abi3-6, fus3-3 (AIMS no. CS8014/N8014), and lec1-2 (AIMS no. CS2922/N2922) showed that protein and HSP17.4::GUS activity were highly reduced in fus3-3 and lec1-2 and undetectable in abi3-6 seeds. In contrast, heat-stressed abi3-6, fus3-3, and lec1-2 seeds stained for GUS activity throughout the embryo. These data indicate that there is distinct developmental and stress regulation of HSP17.4, and imply that ABI3 activates HSP17.4 transcription during development. Quantitation of sHSP protein in desiccation-intolerant seeds of abi3-6, fus3-3, lec1-2, and line24 showed that all had <2% of wild-type HSP17.4 levels. In contrast, the desiccation-tolerant but embryo-defective mutants emb266 (AIMS no. CS3049/N3049) and lec2-1 (AIMS no. CS2728/N2728) had wild-type levels of HSP17.4. These data correlate a reduction in sHSPs with desiccation intolerance and suggest that sHSPs have a general protective role throughout the seed.

Exercise and exhaustion effects on glycogen synthesis pathways.

Female Sprague-Dawley rats were infused with [1-13C]glucose to measure the effect of endurance training and the effect of various metabolic conditions on pathways of hepatic glycogen synthesis. Four metabolic states [sedentary (S), trained (T), sedentary exhausted (SE), and trained exhausted (TE)] were studied. T and TE rats were trained on a motor-driven treadmill (30 m/min, 15% grade, 1.0 h/day, 5 days/wk) for 8-10 wk. After a 24-h fast, SE and TE rats were run to exhaustion (sedentary average = 78 min, trained average = 155 min) at a training pace and immediately infused with labeled glucose for 2 h. S and T rats were infused after a 24-h fast. After infusion, tissues were removed and glycogen was isolated and hydrolyzed to glucose. The glucose was measured for distribution of 13C by using nuclear magnetic resonance. Glycogen was synthesized predominantly by the indirect pathway for all metabolic states, indicating that infused glucose was first metabolized primarily in the peripheral tissue. The direct-pathway utilization was greater in rested S than in rested T animals (30 vs. 14%); however, for exhausted animals, the trained use of the direct pathway was greater (22 vs. 9%). Both TE and rested T animals utilize the indirect pathway a comparable amount. Sedentary animals, on the other hand, dramatically decreased utilization of the direct pathway, with exhaustive exercise changing from 30 to 9%. The results indicate that endurance training modifies glucose utilization during glycogen synthesis after fasting and exhaustive exercise.

Synthesis of small heat-shock proteins is part of the developmental program of late seed maturation.

Small heat-shock proteins (sHSPs) accumulate in plants in response to high-temperature stress. Specific sHSPs, the cytosolic class I and class II proteins, are also expressed in the absence of stress in maturing seeds of several species, and a role for these proteins in desiccation tolerance, dormancy, or germination has been hypothesized. We demonstrate that class I sHSPs are expressed during Arabidopsis seed development in a pattern similar to that previously observed in other species: they are first detected during mid-maturation, are most abundant in dry seeds, and decline rapidly during germination. Although the class I sHSP family in Arabidopsis appears to consist of four genes, expression of a single gene, Athsp 17.4, accounts for the majority of sHSPs in maturing seeds. sHSP levels were also examined in seeds of several Arabidopsis mutants with reduced sensitivity to abscisic acid inhibition, including aba1, abi1, and abi2, abi3-1, abi3-6, abi4, and abi5-1. The abi3-1 mutant has 10-fold reduced levels of sHSPs; sHSPs are undetectable in the abi3-6 mutant. All other mutants were indistinguishable from wild type. These results suggest that sHSP expression in seeds is regulated by the ABI3 response pathway and wild-type levels of sHSPs are not sufficient for seed dormancy and not necessary for desiccation tolerance. However, roles in either process cannot be ruled out. In total the data indicate that the expression of sHSPs in seeds is part of the normal developmental program of late seed maturation and the presence of sHSPs has adaptive significance for plant reproduction.

Determination of the glycogen synthesis pathway by 13C nuclear magnetic resonance analysis.

The level of hepatic glycogen synthesized directly from glucose was measured in rats with [1-13C]glucose. The nuclear magnetic resonance (NMR) spectrum of glucose was used to measure the distribution of the 13C label from C1 to the other carbons. Female Sprague-Dawley rats were surgically implanted with catheters in the left carotid artery and the right jugular vein, followed by a 3-day recovery period and a 24-hour fast to deplete liver glycogen. A 2-hour infusion of the fasted animal with [1-13C]glucose was immediately followed by the removal of blood and liver tissue. The liver was divided into the right, left, caudate, and medial lobes, and then freeze-clamped in liquid nitrogen and stored at -80 degrees C. The 13C NMR glucose spectra were obtained from glycogen that was isolated from each liver lobe and hydrolyzed to glucose with amyloglucosidase. Spectra were obtained at 50.3 MHz in a narrow-bore Gemini 200-MHz NMR spectrometer (Varian, Palo Alto, CA). The distribution of 13C onto glucose carbons was measured from these spectra, and the percent direct pathway was calculated to be 29% +/- 2.5%. Metabolic variation for the synthesis of glycogen within the liver was determined by measuring the direct pathway contribution in each of the four liver lobes. Percent direct pathway values were similar (P > .05) in right (35% +/- 4.9%), left (26% +/- 5.1%), medial (25% +/- 4.9%), and caudate (27% +/- 5.6%) lobes. For some of the animals, the direct pathway was determined by infusion with [6-13C]glucose.(ABSTRACT TRUNCATED AT 250 WORDS)