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ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) of Arabidopsis thaliana is an AP2/ERF domain transcription factor that regulates jasmonate (JA) biosynthesis and is induced by methyl JA treatment. The regulatory mechanism of ORA47 remains unclear. ORA47 is shown to bind to the cis-element (NC/GT)CGNCCA, which is referred to as the O-box, in the promoter of ABI2. We proposed that ORA47 acts as a connection between ABA INSENSITIVE1 (ABI1) and ABI2 and mediates an ABI1-ORA47-ABI2 positive feedback loop. PORA47:ORA47-GFP transgenic plants were used in a chromatin immunoprecipitation (ChIP) assay to show that ORA47 participates in the biosynthesis and/or signaling pathways of nine phytohormones. Specifically, many abscisic acid (ABA) and JA biosynthesis and signaling genes were direct targets of ORA47 under stress conditions. The JA content of the P35S:ORA47-GR lines was highly induced under wounding and moderately induced under water stress relative to that of the wild-type plants. The wounding treatment moderately increased ABA accumulation in the transgenic lines, whereas the water stress treatment repressed the ABA content. ORA47 is proposed to play a role in the biosynthesis of JA and ABA and in regulating the biosynthesis and/or signaling of a suite of phytohormone genes when plants are subjected to wounding and water stress.

SUMMARY Although glutathione is well known for its reactive oxygen species (ROS) scavenging function and plays a protective role in biotic stress, its regulatory function in abiotic stress still remains to be elucidated. Our previous study showed that exogenously applied reduced glutathione (GSH) could improve abiotic stress tolerance in Arabidopsis. Here, we report that endogenously increased GSH also conferred tolerance to drought and salt stress in Arabidopsis. Moreover, both exogenous and endogenous GSH delayed senescence and flowering time. Polysomal profiling results showed that global translation was enhanced after GSH treatment and by the induced increase of GSH level by salt stress. By performing transcriptomic analyses of steady-state and polysome-bound mRNAs in GSH-treated plants, we reveal that GSH has a substantial impact on translation. Translational changes induced by GSH treatment target numerous hormones and stress signaling molecules, which might contribute to the enhanced stress tolerance in GSH-treated plants. Our translatome analysis also revealed that abscisic acid (ABA), auxin and jasmonic acid (JA) biosynthesis, as well as signaling genes, were activated during GSH treatment, which has not been reported in previously published transcriptomic data. Together, our data suggest that the increased glutathione level results in stress tolerance and global translational changes.

ETHYLENE RESPONSE FACTOR1 (ERF1) is an upstream component in both jasmonate (JA) and ethylene (ET) signaling and is involved in pathogen resistance. Accumulating evidence suggests that ERF1 might be related to the salt stress response through ethylene signaling. However, the specific role of ERF1 in abiotic stress and the molecular mechanism underlying the signaling cross talk still need to be elucidated. Here, we report that ERF1 was highly induced by high salinity and drought stress in Arabidopsis (Arabidopsis thaliana). The salt stress induction required both JA and ET signaling but was inhibited by abscisic acid. ERF1-overexpressing lines (35S:ERF1) were more tolerant to drought and salt stress. They also displayed constitutively smaller stomatal aperture and less transpirational water loss. Surprisingly, 35S:ERF1 also showed enhanced heat tolerance and up-regulation of heat tolerance genes compared with the wild type. Several suites of genes activated by JA, drought, salt, and heat were found in microarray analysis of 35S:ERF1. Chromatin immunoprecipitation assays found that ERF1 up-regulates specific suites of genes in response to different abiotic stresses by stress-specific binding to GCC or DRE/CRT. In response to biotic stress, ERF1 bound to GCC boxes but not DRE elements; conversely, under abiotic stress, we observed specific binding of ERF1 to DRE elements. Furthermore, ERF1 bound preferentially to only one among several GCC box or DRE/CRT elements in the promoter region of its target genes. ERF1 plays a positive role in salt, drought, and heat stress tolerance by stress-specific gene regulation, which integrates JA, ET, and abscisic acid signals.

AP2/ERF proteins play crucial roles in plant growth and development and in responses to biotic and abiotic stresses. ETHYLENE RESPONSE FACTOR 53 (AtERF53) belongs to group 1 in the ERF family and is induced in the early hours of dehydration and salt treatment. The functional study of AtERF53 is hampered because its protein expression in Arabidopsis is vulnerable to degradation in overexpressed transgenic lines. Taking advantage of the RING domain ligase1/RING domain ligase2 (rglg1rglg2) double mutant in which the AtERF53 can express stably, we investigate the physiological function of AtERF53. In this study, we demonstrate that expression of AtERF53 in wild-type Arabidopsis was responsive to heat and abscisic acid (ABA) treatment. From results of the cotransfection experiment, we concluded that AtERF53 has positive transactivation activity. Overexpression of AtERF53 in the rglg1rglg2 double mutant conferred better heat-stress tolerance and had resulted in higher endogenous ABA and proline levels compared to rglg1rglg2 double mutants. AtERF53 also has a function to regulate guard-cell movement because the stomatal aperture of AtERF53 overexpressed in rglg1rglg2 double mutant was smaller than that in the rglg1rglg2 double mutant under ABA treatment. In a global gene expression study, we found higher expressions of many stress-related genes, such as DREB1A, COR15A, COR15B, PLC, P5CS1, cpHSC70 s and proline and ABA metabolic-related genes. Furthermore, we identified several downstream target genes of AtERF53 by chromatin immunoprecipitation assay. In conclusion, the genetic, molecular and biochemical result might explain how AtERF53 serving as a transcription factor contributes to abiotic stress tolerance in Arabidopsis.

The relationship between sugar content and loss of desiccation tolerance of hydrated crop seeds (tomato, okra, snow pea, mung bean, and cucumber) was evaluated by imbibing seeds with or without ABA, followed by dehydration and germination. During the process of hydration, but before the seeds lost desiccation tolerance, monosaccharide content increased only slightly, sucrose increased in snow pea, mung bean and cucumber, but maintained its original level in other species and the oligosaccharides declined dramatically. At the time of losing desiccation tolerance, the sucrose content of imbibed seeds was 2-3 times higher than the original level in most species. Positive significant correlation coefficients (r) were found in many, but not all crop seeds between desiccation tolerance and the oligosaccharide mass, or oligo/sucrose ratio. The ratio of oligo/sucrose in intact seeds at the time of losing desiccation tolerance, however, was not a fixed value and varied among species. Oligosaccharides declined significantly in different seed parts of imbibed cucumber seeds while sucrose increased to a higher level in the radicle than in the hypocotyl. Radicles were far more sensitive to desiccation than hypocotyls. The same observation was found for cucumber seeds imbibed in 100 μM ABA, yet desiccation tolerance was largely maintained in hypocotyls and cotyledons. It is concluded that sucrose and oligosaccharides are not the determinants of the loss of desiccation tolerance in hydrated seeds. Imbibed seeds did not show any differences between seed parts in their ability to resynthesize sugars during the process of slow dehydration. Differences in sensitivity to desiccation among seed parts were not due to differences in the initial water content or to the rate of water content increase among seed parts. Physiological regulation of the loss of desiccation tolerance in crop seeds during hydration is discussed.

Seeds of Taxus mairei are known for their deep dormancy which can only be broken by a procedure involving warm stratification followed by cold stratification. Treatments with alternating temperatures of 25}15 or 23}11 ∞C (12 h light) for 6 months followed by 5 ∞C for 3 months were successful in overcoming seed dormancy. After 6 months of warm stratification, cytological changes observed included: enlargement of the embryo; a decrease in the number of lipid bodies; appearance of ER; and increases in mitochondria, plastids, dictyosomes, vacuoles and microbodies in the shoot apical meristem. Cold stratification following the warm treatment induced cell division, and one or two distinct nucleoli in the shoot apical meristem cells were observed. Both warm and cold stratification reduced endogenous ABA concentrations from the original 8888 pg per freshly harvested seed to 392 and 536 pg, respectively. Treatment with exogenous gibberellins after seeds had been warm-stratified showed that GA % and GA ( were eective at promoting seed germination, but GA $ was not. These results suggest that the strong seed dormancy of T. mairei could be caused by a high ABA content and underdevelopment of the embryos in freshly shed seeds. We conclude that warm stratification with alternating temperatures increases the growth of embryos by cell expansion and enlargement and decreases ABA content, but seeds still remain ungerminated. Cold stratification may induce the response to GAs and initiate cell division resulting in release from physiological dormancy and subsequent germination of T. mairei seeds. # 1998 Annals of Botany Company

Biochemical properties, i.e. endogenous abscisic acid, proline, sugars, respiration, adenosine phosphates and adenylate energy charge, and growth and moisture content were measured during the development of seeds of Machilus thunbergii. As dry matter accumulated in the embryo during development, moisture content, ABA, proline, respiration and sugars all declined. At maturity, the dry mass of the seeds failed to attain a plateau before the period of natural seed shedding; the axis and cotyledons reached moisture contents of 58 and 45%, respectively. Drying of immature seeds at 73% relative humidity and 25 °C for 30 d resulted in a complete loss of viability at all developmental stages tested with the exception of mature seeds that were able to tolerate a 5% decrease in moisture content before germination declined. ABA was detected in all embryos tested, with a maximum value 16.·16 μg g -1 d. wt about midway through development. Although the presence of ABA induced no tolerance to desiccation of mature seeds, it did coincide with decreased content of water in the developing seeds and decreased respiration. Desiccation damage of M. thunbergii seeds occurred when moisture content was still high (45%) and this damage was not related to the absence of oligosaccharides in the mature seeds. We conclude that developing embryos and mature seeds of M. thunbergii have properties common to many recalcitrant seeds, with seeds being sensitive to desiccation at all stages, having a prominent ABA peak, little proline, lacking oligosaccharides, and specifically, little dormancy and a moderate rate of respiration of mature seeds (0·9 μmol O 2 min -1 g -1 f. wt). Adenosine triphosphate content and energy charge decreased from stage four to stage eight of seed development, then increased again to 103 nmol g -1 d. wt and 0·73, respectively, in mature seeds. The moderate energy charge observed in mature seeds indicates that continuous metabolism is also a characteristic of recalcitrant seeds.

Fresh mature tree seeds of 16 species plus soybean were used for the analysis of soluble neutral sugar content, as well as for the determination of longevity in terms of the time required for the seeds to decrease to 50% of the original germination under 5 o C and 80-91% relative humidity. Oligosaccharides were sometimes found in species of recalcitrant; although the mass ratio of oligosaccharide/total sugar or mole ratio of stachyose and raffinose/sucrose was less than 0.05 or 0.044, respectively. The time required for seeds to decrease to 50% germination varied from a few days to 8.3 months for the seeds of desiccation-sensitive species. The low ratio of oligosaccharide to sucrose is, however, most unlikely to be a cause of short life-span in recalcitrant seeds

Although glutathione S-transferases (GSTs) are thought to play major roles in oxidative stress metabolism, little is known about the regulatory functions of GSTs. We have reported that Arabidopsis (Arabidopsis thaliana) GLUTATHIONE S-TRANSFERASE U17 (AtGSTU17; At1g10370) participates in light signaling and might modulate various aspects of development by affecting glutathione (GSH) pools via a coordinated regulation with phytochrome A. Here, we provide further evidence to support a negative role of AtGSTU17 in drought and salt stress tolerance. When AtGSTU17 was mutated, plants were more tolerant to drought and salt stresses compared with wild-type plants. In addition, atgstu17 accumulated higher levels of GSH and abscisic acid (ABA) and exhibited hyposensitivity to ABA during seed germination, smaller stomatal apertures, a lower transpiration rate, better development of primary and lateral root systems, and longer vegetative growth. To explore how atgstu17 accumulated higher ABA content, we grew wild-type plants in the solution containing GSH and found that they accumulated ABA to a higher extent than plants grown in the absence of GSH, and they also exhibited the atgstu17 phenotypes. Wild-type plants treated with GSH also demonstrated more tolerance to drought and salt stresses. Furthermore, the effect of GSH on root patterning and drought tolerance was confirmed by growing the atgstu17 in solution containing l-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH biosynthesis. In conclusion, the atgstu17 phenotype can be explained by the combined effect of GSH and ABA. We propose a role of AtGSTU17 in adaptive responses to drought and salt stresses by functioning as a negative component of stress-mediated signal transduction pathways.

Mutants of Arabidopsis thaliana (L.) Heynh. with altered regulation of starch degradation were identified by screening for plants that retained high levels of leaf starch after a period of extended darkness. The mutant phenotype was also expressed in seeds, flowers, and roots, indicating that the same pathway of starch degradation is used in these tissues. In many respects, the physiological consequences of the mutations were equivalent to the effects observed in previously characterized mutants of Arabidopsis that are unable to synthesize starch. One mutant line, which was characterized in detail, had normal levels of activity of the starch degradative enzymes alpha-amylase, beta-amylase, phosphorylase, D-enzyme, and debranching enzyme. Thus, it was not possible to establish a biochemical basis for the phenotype, which was due to a recessive mutation at a locus designated sex1 at position 12.2 on chromosome 1. This raises the possibility that hitherto unidentified factors, altered by the mutation, play a key role in regulating or catalyzing starch degradation.

Selaginella tamariscina, one of the most primitive vascular plants, can remain alive in a desiccated state and resurrect when water becomes available. To evaluate the nature of desiccation tolerance in this plant, we compared the composition of soluble sugars and saturation ratios of phospholipids (PLs) between hydrated and desiccated tissues of S. tamariscina using gas chromatography. In this study, differences in gene expression and ABA contents were also analyzed during dehydration. The results revealed that trehalose (at >130 mg g(-1) DW) was the major soluble sugar, and low saturated fatty acid content in PLs (0.31) was maintained in both hydrated and desiccated tissues. In addition, the ABA content of S. tamariscina increased 3-fold, and genes involved in ABA signaling and cellular protection were up-regulated while photosystem-related genes were down-regulated during dehydration. The biochemical and molecular findings suggest that both constitutive and inducible protective molecules contribute to desiccation tolerance of S. tamariscina.

A new cyclitol which is abundant in the late developmental stages of leucaena (Leucaena leucocephala (Lam.) de Wit) seeds was identified by HPLC, NMR, and GCMS as O-a-D-gaIactopyranosyl-(l —*• l)-3-O-methyl-D-cA/roinositol, a new galactopinitol. This galactopinitol was initially detected midway through seed development and increased to 10.2 mg (g DW)~\ but decreased in mature seeds to its about a half. Stachyose content increased greatly and remained the most abundant of the soluble sugars in mature seeds (25.6 mg (g DW)' 1 ). Artificial drying at 73% relative humidity of 70 DPA immature seeds induced the accumulation of raffinose, stachyose, galactopinitol and galactinol, but the total amounts of these sugars were only about half of those found in mature seeds. Seed germination decreased following an initial increase after 8 d artificial drying to a moisture content of 24%, and this dehydration damage probably is because of underdevelopm ent of seed tissue. Galactopinitol changes in a similar fashion to the oligosaccharides during the late developmental stage and dehydration experiment, implying that galactopinitol may play a role in desiccation tolerance of leucaena seeds.

A starch deficient mutant of Arabidopsis thaliana (L.) Heynh. has been isolated in which leaf extracts contain only about 5% as much activity of ADPglucose pyrophosphorylase (EC 2.7.7.27) as the wild type. A single, nuclear mutation at a previously undescribed locus designated adg2 is responsible for the mutant phenotype. Although the mutant contained only 5% as much ADPglucose pyrophosphorylase activity as the wild type, it accumulated 40% as much starch when grown in a 12 hour photoperiod. The mutant also contained about 40% as much starch as the wild type when grown in continuous light, suggesting that the rate of synthesis regulates its steady state accumulation. Immunological analysis of leaf extracts using antibodies against the spinach 54 and 51 kilodalton (kD) ADPglucose pyrophosphorylase subunits indicated that the mutant is deficient in a cross-reactive 54 kD polypeptide and has only about 4% as much as the wild type of a cross-reactive 51 kD polypeptide. This result and genetic studies suggested that adg2 is a structural gene which codes for the 54 kD polypeptide, and provides the first functional evidence that the 54 kD polypeptide is a required component of the native ADPglucose pyrophosphorylase enzyme.

Pectinmethylesterase from the pericarp of jelly fig (Ficus awkeotsang) achenes was extracted and purified to a specific activity of 289 micromole proton produced per minute per milligram protein. Pectinmethylesterase, a major protein with high specific activity in the crude extract, was monomeric with a molecular weight of 38,000. The enzyme preparation was stable in distilled water at 4 degrees C for at least 6 months, and at 60 degrees C for at least 10 minutes. This enzyme functioned optimally at pH 6.5 to 7.5 when the assay mixture contained no NaCl or at low NaCl concentration. The pH optimum shifted to lower pH as the NaCl concentration was increased. The K(m) value for pectin was 0.75 milligram per milliliter pectin, corresponding to a V(max) value of 310 micromoles per minute per milligram protein. Inhibition studies with antibodies indicated that jelly fig achene pectinmethylesterase and the two other pectinmethylesterases from orange and tomato were similar in their active site conformation; however, the surface determinants may be very different because no precipitation between anti-jelly fig pectinmethylesterase immune serum and the pectin methylesterase from orange and tomato could be observed in the double immunodiffusion analysis. Specific antisera raised against jelly fig achene pectinmethylesterase in a Western blot experiment also showed low similarity between jelly fig pectinmethylesterase with that from orange and tomato. This observation was also supported by the very low isoelectric point (pH 3.5) of jelly fig pectinmethylesterase, compared with high isoelectric points reported for most of the pectinmethylesterases. Amino acid composition and N-terminal sequence have been obtained. High homology of the N-terminal amino acid residues between jelly fig and tomato pectinmethylesterase (O Markovic, H Jornvall [1986] Eur J Biochem 158: 455-462) was observed. Pectinmethylesterase activity causes the release of protons from the deesterification of pectin such that a low pH environment is created, and this may be related to the cell growth. Pectinmethylesterase is not needed for jelly fig seed germination, however the gel formed from pectin and pectinmethylesterase may insure a water source for the germinating jelly fig seeds.

Three classes of mutants of Arabidopsis thaliana (L.) Heynhold with alterations in starch metabolism were found to have higher levels of leaf amylase activity than the wild type when grown in a 12-hr photoperiod. This effect was dependent upon the developmental stage of the plants and was largely suppressed during growth in continuous light. The various amylolytic activities in crude extracts were separated by electrophoresis in nondenaturing polyacrylamide gels and visualized by activity staining. The increased amylase activity in the mutants was due to an up to 40-fold increase in the activity of an extrachloroplast β-amylase (EC 3.2.1.2). These observations indicate the existence of a regulatory mechanism that controls the amount of β-amylase activity in response to fluctuations in photosynthetic carbohydrate metabolism. It is paradoxical that β-amylase appears to be a highly regulated enzyme, but as yet no physiologically relevant function can be assigned to this enzyme due to the absence of starch in the cytoplasmic compartment of leaf cells.

A major triticale (X Triticosecale Wittmack) endosperm acid phosphatase (EC 3.1.2.2) (APase) from sib-lines producing plump and shriveled seed was purified 140- and 230-fold to a specific activity of 94 and 153 micromoles per minute per milligram protein respectively, by ammonium sulfate fractionation, ion-exchange chromatography, chromatofocusing, affinity column chromatography, and gel filtration. The purified enzyme from both materials is a monomeric glycoprotein with an apparent molecular weight of 45,700 +/- 500 containing 12% carbohydrate and an apparent isoelectric point of pH 5.9. It hydrolyzes tri- and di-phosphate of nucleosides as well as phosphate esters and exhibits characteristics of ATP-hydrolase and phosphatase. About 2-fold more of the APase was isolated from shriveled seeds, and the purified enzyme exhibited 3- and 5-fold higher V(max) for p-nitrophenyl phosphate and ATP, respectively, than that of plump seed. The I(50) for Pi concentration was 5.5-fold higher in APase of shriveled seed than the plump one. These varied quantitative and kinetic properties substantiate the role of APase in lines with shriveled seeds being reduction of starch accumulation by depleting substrates and energy supply in the cytosol.

Amylolytic enzymes of Arabidopsis leaf tissue were partially purified and characterized. Endoamylase, starch phosphorylase, d-enzyme (transglycosylase), and possibly exoamylase were found in the chloroplasts. Endoamylase, fraction A2, found only in the chloroplast, was resolved from the exoamylases by chromatography on a Mono Q column and migrated with an R(F) of 0.44 on 7% polyacrylamide gel electrophoresis. Exoamylase fraction, A1, has an R(F) of 0.23 on the polyacrylamide gel. Viscometric analysis showed that A1 has a slope of 0.013, which is same as that of A3, the extrachloroplastic amylase. A1, however, can be distinguished from A3 by having much higher amylolytic activity in succinate buffer than acetate buffer, and having much less reactivity with amylose. A1 probably is also localized in the chloroplast, and contributes to the 30 to 40% higher amylolytic activity of the chloroplast preparation in succinate than acetate buffer at pH 6.0. The high activity of d-enzyme compared to the amylolytic activity in the chloroplast suggests that transglycosylation probably has an important role during starch degradation in Arabidopsis leaf. Extrachloroplastic amylase, A3, has an R(F) of 0.55 on 7% electrophoretic gel and constitutes 80% of the total leaf amylolytic activity. The results of substrate specificity studies, action pattern and viscometric analyses indicate that the extrachloroplastic amylases are exolytic.

A certain amount of the carbon dioxide that is converted to 3-P-glycerate in plants during photosynthesis is utilized for formation of the reserve polysaccharides present in the leaf starch granule. However, the fraction of carbon accumulated as starch in different plants varies. Species such as barley, spinach and wheat accumulate proportionately more sucrose than starch whereas the leaves of peanuts, soybean and tobacco accumulate more starch than sucrose. It is believed that an important factor in the partitioning of carbon between sucrose and starch is the level of sucrose-P synthase (1,2). The levels of sucrose-P synthase in the leaf cell correlates fairly well with the amount of 14CO2 incorporated into sucrose and inversely correlates with the amount of 14CO2 fixed into the starch fraction.

Two major forms of d-enzyme (4-alpha-glucanotransferase, EC 2.4.1.25) were successfully separated from most of the amylase activity using FPLC-Mono Q column chromatography. Transfer of a maltosyl group was observed upon the incubation of d-enzyme with maltotriose and d-[U-(14) C]glucose. About 4.5% of the radioactivity was transferred to maltotriose in 2 hours. End product analysis showed the accumulation of glucose and maltopentaose from maltotriose within the first 10 minutes of the reaction. Several other maltodextrins were also observed with longer incubation times, although maltose was never produced. A quantitative measurement of maltodextrin production from the reaction of [(14) C]maltotriose with d-enzyme showed that the quantity of maltotriose decreased from 100% to 31% after 3 hours incubation, while glucose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, and higher maltodextrins increased in amount. Glucose is the major product throughout the course of the reaction of d-enzyme with maltotriose. Maltotriose, in addition to glucose, are the major products in the reaction of d-enzyme with maltodextrins with a chain length greater than maltotriose. This study confirms the existence of a transglycosylase that disproportionates maltotriose and higher maltodextrins by transferring maltosyl or maltodextrinyl groups between maltodextrins resulting in the production of glucose and different maltodextrins, but not maltose, in leaf tissue with enzymic properties very similar to the previously reported d-enzyme in potato.