Ca2+ levels, was studied in tomato plants grown in

559 CHARACTERIZATION OF PHOSPHATE STARVATION INDUCED GENE EXPRESSION IN THE ROOTS OF TOMATO CHUNMING LIU. Umesh S. Muchhal. Genyi Zhang and K.G. Ragh.othama, Department of Horticulture, Purdue University, West Lafayette, IN. 47906. Phosphate starvation could lead to several morphological and biochemical changes in the roots of plants. Some of these changes have been shown to involve altered gene expression. We are interested in understanding the changes occurring at the level of gene expression during the early periods of phosphate starvation. Aeroponically and hydroponically grown tomato plants were subjected to Pi stress (0 Pi) for various periods of time. The mRNA isolated from Pi starved tomato roots were used for preparing cDNA libraries in .Zap vectors. Several partial cDNA clones representing Pi starvation induced mRNA in roots were isolated by the technique of differential display of RNA species. One of the cDNA (pPIG1, Ehosphate starvation Induced flene) hybridized strongly to a mRNA from Pi starved roots of tomato. Expression of PIG1 is induced within 24 h of Pi starvation, and increases several fold with continued starvation. The PIG1 is expressed in both roots and leaves of Pi starved tomato, whereas its expression is much higher in roots. A full length cDNA clone was obtained from the cDNA library, using a PM 1 specific 3' primer and a T3 primer by polymerase chain reaction. This clone is 474 bp in length and hybridizes to a mRNA, approximately 500 bp long. A comparison of the nucleotide and the deduced amino acid sequence of the longest open reading frame of PIG01 with nucleotide and protein sequence data bases indicated that it does not have a high degree of identity with any known gene sequences. The significance of Pi starvation on gene expression in roots will be discussed. This research was supported, in part, by USDAINRICGP grant # 94-37100-0834.

560

IDENTIFICATION OF THE cDNA THAT CORRESPONDS TO A 5 kD Zn-BINDING PROTEIN IN CITRUS BLIGHT KATHRYN C. TAYLOR and Manasa Ravindranath Department of Plant Sciences, University of Arizona, Tucson, AZ 85721. The level of a 5 kD Zn-binding protein is increased in association with a disorder in citrus, citrus blight. The protein appears responsible for the sequestration of Zn in the phloem tissue with the loss of Zn from other tissues in the tree. The protein was purified to homogeneity and a N-terminal amino acid sequence was determined. The sequence indicates that the protein is cysteine-rich, with two cys-x-cys metal binding domains evident. Degenerate oligonucleotide primers were synthesized based on this sequence. PCR has yielded products that were subcloned. These were used for screening leaf and phloem specific cDNA libraries. Sequencing of clones is underway. These studies were funded by the Arizona Citrus Research Council: ACRC#94-7.

562 CHARACTERIZATION OF ARABIDOPSIS RESPONSE TO LOW P MELANIE C. TRULL1, Mark J. Guiltinan 12, Jonathan P. Lynch2, M1 Deikman- 1,3 . 1 Biotech. Institute, 2 Hort. Dept., 3 Biol. Dept., Penn. State Univ., University Park, PA 16802 Phosphorus availability in native soils is seldom adequate for optimal plant growth. We are characterizing the response to low P in Arabidopsis so that

we can use molecular genetics to help elucidate the mechanism plants have of sensing and responding to P starvation. We have found that Arabidopsis plants under low P conditions accumulate anthocyanins, the root to shoot ratio increases, and acid phosphatase activity in the roots increases approximately two-fold. Acid phosphatase can solubilize organic phosphates in the soil, increasing the availability of P. We are examining the activity of different acid phosphatase isozymes under low and high P conditions. We have isolated two mutants that apparently fail to secrete acid phosphatase in response to low P. These mutants could be defective in detecting low P conditions, in producing acid phosphatase, or in secreting acid phosphatase. We are currently characterizing these mutants to determine which aspect of their response is defective. Supported by the DOE/NSF/USDA CRPB program (NSF grant BIR-9220330).

563 PHOSPHATE STARVATION INDUCED CHANGES IN THE EXPRESSION OF TOMATO Ca2+-ATPase. UMESH S. MUCHHAL and K.G. Raghothama Department of Horticulture, Purdue University, West Lafayette, IN-479071165 The Pi deficiency results in retarded plant growth and severe reduction in yield. Plants have developed adaptive mechanisms such as production and secretion of enzymes and organic acids, and modification of root architecture and ion transport mechanism to enhance both the availability and uptake of Pi upon starvation. We are investigating the molecular mechanisms underlying the response of plants to Pi starvation. The modulation of intracellular Cau levels has been proposed to be a major component of many signal transduction pathways in plants. The expression of Ca2i-ATPase, which plays a significant role in modulation of cytosolic Ca2+ levels, was studied in tomato plants grown in aeroponics and subjected to Pi starvation. The total RNA from these plants was isolated and analyzed by northern blotting using a 0.9 Kb EcoRI fragment of a cDNA clone (LCA, kindly provided by Dr. Allen Bennett, UC-Davis) encoding tomato Ca2+ -ATPase. The levels of Ca *-ATPase transcript increased in the roots with the increasing duration of Pi starvation. The initial response was apparent after 24 hrs of starvation and reached its maximum of 5-10 fold increase after 7 days. The levels of Ca2+-ATPase transcript remained more or less constant in leaves during this period of Pi starvation. The expression of H+-ATPase did not change significantly during this period in both roots and leaves. The implications of changes in cytosolic CaJ-levels during Pi starvation are discussed.

564 561 EFFECT OF PHOSPHORUS AVAILABILITY ON BASAL ROOT GROWTH ANGLE IN BEAN AMY M. BONSER. lonathan Lynch & Sieglinde Snapp. Dept. of Horticulture, The Pennsylvania State University, University Park, PA 16802 Phosphorus deficiency is a major constraint to plant growth in most terrestrial ecosystems. Root architecture may be of particular importance for P acquisition because P is very immobile in soil. Gravitropism, lateral branching, and root growth rate all contribute to root architecture; therefore, each of these aspects of root architecture may be important for root adaptation to low P soils. Because P is generally richer in upper soil horizons, root gravitropism may influence P acquisition in these upper horizons. We examined the effect of low P availability on root gravitropism by measuring root growth angles. Sixteen bean genotypes were grown with and without P in growth pouches, and growth angles of basal roots were measured over time. For some genotypes, basal roots had more shallow growth angles when grown without P, while others had deeper angles when grown without P, and others showed no difference in growth angle. Genotypes with shallow growth angles had better seed yields on low P soils, suggesting that this response may be an adaptation to low P soils. Using P buffered sand culture, the response in the growth pouches was confirmed in 3-D. The effect of P on growth angle was found to be to local P availability using a split pouch system and to be specific to P. The changes in growth angle with respect to P treatment occurred before differences in P content of plant tissues were found and before growth differences between treatments occurred. For these reasons, we suggest that these early root responses of bean to low P may be to external P availability and not due to P deficiency of plant tissue. This research was supported by the DOE/NSF/USDA CRPB program (NSF BIR-922-330).

112

IN POTATO LEAVES BIOTIC AND ABIOTIC STRESSES INDUCE THE EXPRESSION OF ACC SYNTHASE mRNA ALSO INDUCED BY

OZONE Carl D. Schlagnhauferl RICHARD N. ARTECAL. & Eva J.PeLll Dept. of Horticulture; 2Dept. of Plant Pathology; The Pennsylvania State University, University Park, PA 16802 Ethylene production is involved in many plant physiological processes including stress responses and is frequently associated with foliar senescence. Ethylene emission is a common plant response to many biotic and abiotic stresses. We have previously cloned a cDNA (QIP-I) for an ozone-induced ACC synthase in leaves of Solanum tuberosum L. Experiments were conducted to deternine whether the OIP-1 gene would be expressed in potato foliage treated with other stresses which induced ethylene. We selected two abiotic stresses, CuCl2 and sorbitol, an osmotic stress, and two fungal pathogens, Alternaria solani Sorauer, causal agent of early blight, and Phytophthora infestans (Mont) de Bary, causal agent of late blight. When excised leaves were treated with 5 mM CuCl2 ethylene production increased within 2h and mRNA levels of OIP-1 increased concomitantly. Similar results occurred when excised leaves were treated with 2 M sorbitol. When intact plants were inoculated with either of the two fungal pathogens, ethylene emission began 2-3 days after inoculation, prior to the appearance of significant symptoms. Ethylene emission continued to increase for the 4 days of the experiment. The mRNA levels of OIP-1 increased, but were significantly lower than levels detected for comparable levels of ethylene induced by ozone. The degree of expression of OIP-1 in response to both biotic and abiotic stress and its relationship with ethylene production and senescence in potato leaves will be discussed. This study was supported in part by NRICGP 93-37100-8921.

565

568

THE EFFECTS OF LONG-TERM CO2 ENRICHMENT ON PHOTOSYNTHESIS, STOMATAL CONDUCTANCE AND INTERNAL/EXTERNAL C02 CONCENTRATIONS IN PINUS PONDEROSA. MATTHEW P. TORRES1, James L.J. Houpis1, James C. Pushnik 2 ' Health and Ecological Assessment Division, Lawrence Livermorc Nat. Lab, Livermore, CA 94550; 2Dept. Bio. Sci., Cal St. Chico, Chico, CA 95929 Today's ambient CO2 level is expected to increase two-fold (-350 umol/L), by the middle of the 2l1' century. Higher levels of CO2 are expected to cause major changes in the morphological, physiological, and biochemical activities of the world's vegetation. Therefore, we constructed an experiment which monitored the long-term acclimation processes of Pinus Ponderosa. As a prominent forest conifer, Pinus Ponderosa has a large impact on the carbon cycle. Eighteen genetically variable families were exposed to 3 different levels of CO2 (350 umol/L, 525 umonL, 700 umoL), for two growing seasons (April 1992 to August 1994). Using a LICOR Ll6250, photosynthetic rate, stomatal conductance and internal external CO2 measurements were taken. Photosynthesis increased dramatically over increased CO2 levels (-1.8 umol mr2 sl ). Within each CO2 level, half-sibling families tended to have lowver photosynthetic rates than open pollinated families. Stomatal conductance increased significantly by approximately 0.006 mol m2s7l (p < .05), with increasing CO2-. Conductance values across tree variability, however, shifted very little. Internal CO2 concentrations within the needles increased relatively to the external CO2 concentrations. Ci/Ca values rose from 64.94/o in ambient (350 umol/L), chambers to 68.6% in enriched chambers (700 umol/L). Generally, half-siblings, rather than open pollinated species, contained greater amounts of internal CO2 when external CO2 increased Assuming it can withstand the stress of increasing atmospheric CO2, Pinus Ponderosa may serve as an effective global carbon sink in the future. This research was funded by the United States Department of Energy and the Califomia State University system.

OVERPRODUCTION OF Cu/Zn-SUPEROXIDE DISMUTASE IN THE CYTOSOL OF TOBACCO PROVIDES PARTIAL OZONE TOLERANCE LYNNE H. PITCRER, Eugene P. Radar, & Barbara A. ZilinYkas, Plant Science Dept., Cook College, Rutgers Univ., New Brunswick, NJ 08903 Ozone damage is mediated by the action of toxic oxygen species against which cellular antioxidant systems are a front-line defense. Cytosolic

566 DOES OVERPRODUCTION OF ASCORBATE PEROXIDASE IN THE CHLOROPLAST INFLUENCE OZONE-INDUCED ALTERATIONS OF PHOTOSYNTHESIS? GRO TORSETHAUGEN 1, L H. Pitcher 2, Barbara A. Zilinska 2 & Eva J. EIL3 1Department of Biology, P.O.Box 1045, University of Oslo, Norway. 2Plant Science Department, Rutgers University, New Brunswick, NJ 08903. 3Department of Plant Pathology and Environmental Resources Research Institute and The Pennsylvania State University, University Park, PA 16802. Ozone (03) has been shown to reduce photosynthesis and accelerate foliar senescence. These phenomena are associated with an increase in degradation of ribulosel,5-bisphosphate carboxylase oxygenase (Rubisco) and decline in mRNA for rbcS and rbcL. It is not known whether 03 induces these changes through direct oxidative events in the chloroplast. or indirectly through signals evoked by extrachloroplastic events. Transgenic tobacco plants (Nicotiana tabacwn cv Bel W3) have been developed which overproduce the pea (cytosolic) ascorbate peroxidase (APX) in the chloroplasts (Supplement to Plant Physiol.105:1 16). We conducted experiments to determine whether the presence of increased antioxidant potential in the chloroplasts could protect against 03induced reduction in photosynthesis. Transgenic and nontransgenic plants, grown in a charcoal-filtered greenhouse, were exposed to 0.08 g11-1 in continuous stirred tank reactors from 0900 to 1300 h for 7 days. On the first day of exposure the most recently matured leaf was tagged on each plant. Each day of exposure and for the 5 subsequent days net photosynthesis was measured by gas exchange analysis. Tagged leaves were then harvested and ethylene emission was determined. Subsamples were frozen for subsequent analysis of APX activity, Rubisco quantity and levels of rbcS and rbcL mRNA. Physiological and biochemical measurements were correlated with occurrence of visual injury and signs of accelerated senescence. This work was funded in part by EPA Grant No.

R820010-01-0.

superoxide dismutase (SOD), one such antioxidant, has been shown to increase in response to ozone. The current work tests the hypothesis that plants overexpressing cytosolic SOD are more tolerant to ozone. Pea cytosolic Cu/Zn-SOD under the control of the CaMV35S promoter was overexpressed by 1.5to 3-fold in the cytosol of each of two varieties of tobacco: Wisconsin 38 (W38) and Bel W3, a particularly sensitive ozone variety. SOD overproducing progeny of both cultivars were exposed to acute ozone doses in several separate experiments and non-senescent leaves were scored for visible necrosis. Two independent W38 transgenic lines were tested and both showed fairly uniform reduction of necrosis, by about half, on all rated leaves as compared to their non-transformed controls. All four Bel W3 tranagenic lines tested showed protection on young leaves; however, in older leaves the response was variable. These results demonstrate that Cu/Zn-SOD in the cytosol can function in reducing ozone-induced necrosis. EPA grant R820010-01-0 supported this research. 569 A

GI.UTATHIIONE REDUCTASE ISOIFORM IN ISOI.A1 ED PEA (I)isum salivum

L.) PROTOI'IIASTS CIIANGES IN RESPONSE TO OXIDATIVE STRESS. CAMEILIA MOSES OKPOI)U and Ruth 0. Alscher Dept of P'lant lPathology. titysiology and Weed Science Virginia Polytcchnic Institute and Stite University, Blacksburg, VA 24061-0331 The relative responses of CO, -dependent 0,-evolution and glutatliione reductase (GR)

activities in the presence of oxidative stress (10 mM sodium sulfite) were studied in two cultivars of pea, Progress and Nugget In Progress the CO,-dependent 0,evolution rates dropped by SO/ after I hour and continued to decline (>90%/.) for up to 2 hours. As determined spectrophotometrically, OR activity was unchanged in protoplasts from Progress over the entire 2 hour time course. In contrast, in Nugget protoplasts, exposure to 10 mM sulfite for hour resulted in a 60f/h decrease in COdependent 02-evolution after which rates returned to control levels. The GR activity had increased by 30/O (at the end of I hour) and by the 2 hour time point it had returned to control levels. In both cultivars, only a single polypeptide band corresponding to GR activity was detected via one dimensional native activity gels. Sulfite treated protoplasts isolated from Progress showed no significant difference in GR activity as determined by densitometer scans of the native activity gels. However, sulfite treated Nugget protoplasts showed an increase in GR activity at 30 minutes after which the GR activity returned to control levels These results were consistent with the spectrophotomctric assay results When the protein extracts from both cultivars were further resolved on native analytical IEF, distinct isoforms of GR were detected using a GR activity stain. In Progress protoplasts, two forms of the enzyme were resolved (pl 4.0 and 3 1) and the relative abundance of the isoforms did not change in reponse to sulfite stss. However, in Nugget protoplasts three isoforns (p1 4.0, 4.3 and 5.1) were resolved. The isoform with a p1 of 4.3 increased after treatment with 10 mM sulfite. Our results suggest that a stress-inducible form of OR is present in Nugget protoplasts. Future work is underway to determine if this stressinduced isoforn is due to a different gene product, or if it is the result of posttranscriptional or post-translational modifications of a single gene product.

567

OZONE-INDUCED SHIFTS IN RUBISCO QUANTITY AND SYNTHESIS AT VARYING POSITIONS WITHIN THE CANOPY OF HYBRID POPLAR SAPLINGS BRYAN W. BRENDLEYI and Eva 1. Pell2 1Graduate Program in Plant Physiology and 2Departsnent of Plant Pathology and Environmental Resources Research Institute, The Pennsylvania State University., University Park, PA 16802 In experiments conducted in 1993 we confirmed that older foliage of ozone (03)-stressed hybrid poplar trees (Populus maximowizii x trichocarpa clone 245) exhibited a reduction in quantity of ribulose bisphosphate carboxylase/oxygenase (Rubisco). This reduction was presumed to be due to an increase in protein degradation, as protein labeling experiments showed no difference in rates of protein synthesis in charcoal filtered versus 03-treated foliage. Foliage from 03-treated plants, at a higher position in the canopy, showed a transient increase in Rubisco quantity as well as an increase synthesis of the protein. Experiments conducted in 1994 were designed to develop an understanding of the relationship between degradation of Rubisco (due to acCelerated senescence) and subsequent synthesis of the protein at different locations in the canopy. Using a 1% Phorwite Bru (Miles Inc.) solution we detennined that every fourth leaf on this hybrid poplar clone was connected directly by major leaf traces. Plants were grown in open-top chambers from June until Scptember 1994 and treated with 0.08 ul 1-1 03 from 1000-1800 h daily. Eight times in an 133 day interval every fourth leaf was analyzed for photosynthetic rate, and for quantity and degree of synthesis of Rubisco. The greatest adverse effects of 03 were observed on the oldest harvested leaf. The leaf traced to this older leaf showed an accelerated increase in the synthesis of Rubisco. In addition, we observed that leaf initiation occurred more rapidly in 03-treated saplings. The differences in timing of leaf initiation and synthesis of Rubisco in 03-treated hybrid poplar saplings may reflect a compensatory strategy to the adverse effects of this stress.

570 METABOLIC RESPONSES OF LUFFA ROOTS TO LONG-TERM FLOODING Pai Hsiang Su& CHIN HOLIN Botany Dept.,Natl. Chung Hsing Univ., Taichung, Taiwan, Rep. of China Luffa (Luffa cylitidrica Roem. cv. Cylinder #2), a ilood-toierant species, can accommodate itself to flooding in nature, caused by prolonged monsoon rain. In the present paper, metabolic responses of luffa roots to experimental flooding were evaluated. Upon flooding, the respiratory capacity of flooded roots decreased to 60% of the nonflooded level. The activity of pyruvate decarboxylase (PDC, EC 4.1.1.1) and alcohol dehydrogenase (ADH, EC 1.1.1.1) increased rapidly and steadily. After 7 days of flooding, almost SO-fold increase of both enzymes indicated that strong ethanolic fermentation capacity was induced in response to long-term flooding. In contrast to PDC and ADH activity, ethanol level showed no substantial increase, but declined after an initial rise at day one. Noticeably, a high ADH/PDC ratio in activity may be of importance to avoid accumulation of toxic levels of acetaldehyde. In the other fermentative pathways, the activity of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the key enzyme catalyzing malate synthesis, showed no measurable difference between nonflooded and flooded roots. However, NADP-malic enzyme (NADP-ME, EC 1.1.1.40), catalyzing the decarboxylation of malate, exhibited a markedly increased activity in flooded roots as compared with nonflooded controls. Moreover the level of malate in flooded roots dropped to 40% of the control level. These results are inconsistent with a current metabolic theory of flooding tolerance. The activity of lactate dehydrogenase (LDH, EC 1.1.1.27)slightly rose in response to flooding, and an initial rise of lactate was observed in flooded roots. The activity of two key enzymes for the regulation of glycolysis: phosphofructokinase (PFK, EC 2.7.1.11) and pyruvate kinase (PK, EC 2.7.1.40)increased in flooded roots to 1.S-and 4-fold of the control level respectively, so that the increase in glycolytic capacity appears regulated by "coarse' control in flooded roots. This research was supported by grant NSC-83-021 1-B-005-076 from the National Science Council, Republic of China.

113

571

574

EFFECT OF ABSCISIC ACID ON PARAHELIOTROPIC LEAF MOVEMENT IN PHASEOLUS Eric Lyons & VIRGINIA S. BERG, Biology Department, University of Northern Iowa, Cedar Fall, IA 50614-0421 The motor cells that tip leaves of Phaseolus upward during hot, bright, dry conditions are similar to guard cells in many respects, but much less is known about the biochemical control of these paraheliotropic (lightavoiding) leaf movements. While abscisic acid (ABA) is known to be involved in opening ion channels in guard cell membranes, the effects of this compound on the motor cells controlling stress-associated leaf movements has not been determined. Intact plants growing in a greenhouse were irrigated with ABA solutions (10-i M) or with water. Stomatal conductance was measured using a porometer, and leaf angle above horizontal was determined with an inclinometer. Measurements were taken for several hours after treatment; at the final measurement, water potential was determined with a pressure chamber. Plants treated with ABA had lower conductances, higher water potentials and higher leaf angles than control plants treated with water. The effects on leaf angle were not apparent until well after stomates had responded. Leaves treated with ABA had high water potentials, but were maintained at angles normally seen only in water stressed plants. Thus ABA can substitute for low water potential in promoting paraheliotropic leaf movements.

ELICITOR-INDUCED ACTIVATION OF A TOBACCO SESQUITERPENE CYCLASEPROMOTER. SHAOHUIYIN JoshChapp. AgronomyDept., Univ. of Kentucky, Lexington, KY 40506

5-epi-aristoochene synthase (EAS), a sesqukterpene cyclase, calalyzes a key regulatory step in the synthesis of sesqusterpene phytoalexins that is characteristic of solanaceous plants. The expression of EAS genes is absent from control or non-treated plant tissues, but rapidly induced to high lvels upon pathogen or elicitor treatment. Previous studies showed that the nduction of EAS enzyme activity required transcription of the correspodng gene. Regulatory properties of a tobacco EAS gene promoter were therefore investigated in transgenic tobacco containing an EAS-GUS fusion. Promoter activiy was monitored by histochemical and quantiative assay of GUS activity. Elicitins (fungal proteins eliciting plant defense responses) induced the expression of the gene fusion in transgenic tobacco leaves and stems. The induced GUS activity was restricted to tissue in the immediate vicinity of iocalized treatment with elicitins. As with endogenous sesquiterpene cyciase, the gene fusion was not induced by leaf injection with 5 mM H202 or 0.5 mM SA, suggesting that the signal transduction pathway involved in capsikdol phytoalexin accumulation is different from that responsble for the activation of other PR genes. Analysis of '-deletions of the EAS4 promoter (oss of function) as well as fusion of selected promoter sequences to a minimal CaMV 35S promoter (-90 to +8) (gain of function) are currently being used to identify and fine map those sequences within the promoter responsible for elickitor-inducible gene expression. This work is suppored by the NSF.

572

575

MAIE INDUCTION OF LDH OCCURS IN THE ROOT AXS, AND NOT THE ROOT TIP UNDER LONG TERM EXPOSURE TO HYPOXIA DAVID M. MACALPINE David L. Andrew Malcolm C. Drew. and B.Gr Cobb Department of Hort. Science, Texas A&M University, College Station, TX 77843 The ability of a plant to survive flooded conditions may depend, in part, on energy produced anaerobically during fermentation to ethanol or lactic acid. The DaviesRoberta hypothesis prodicts only a transient formation of lactic acid in anoxic maize root tips, catalyzed by lactate dehydrogenase (LDH), prior to sustained ethanolic fermentation, and suggests that lactic acid production contrbutes to cytoplasmic acidosis. However, barley roots, when deprived of oxygen, exhibit a long term induction of LDH, which might indicate an alternative role for LDH in oxygen-depleted tissues. We examined LDH actrivty in maize seedlings exposed to long term oxygen stress in a variety of tissues (pnmary root axis, nodal root axis, prmasy root tip, and the nodal root tip) from seedlings of different ages exposed to hypoxic and anoxic conditions. Oxygendeprivation was brought about using either an 'open system" in which shoots were exposed to air (21% v/ 02) and the roots were in a solution sparged with 02-free N2, or a "closed system" in which both the shoots and roots were in the same enclosed environent. Results showed that there was no increase in LDH transcripts or enzymatic activity in the root tips under any conditions of 02deprivation in either the open or closed systems. Additionally, in the 'closed system" no significant induction occurred in any tissue. In both prinasy and nodal root tips LDH transcript levels remained constant and at the same values as in the aerobic controls, the LDH enymatic activity in this tissue also remained constant at approximately I Ulgfw regardiess of the system. However, in both prinasy and nodal root axes, transcipt levels were increased appreciatively in the open system. LDH activity in the root axes started at less than 0.1 Ulgfw and was incrsed 4-5 fold at 72h. A range of hypoxic and anoxic treatments from 0%-4% 02 was applied in the closed system in an attempt to duplicate the induction evident in seedlings treated in the open system, where there is preumably a gradient of 02 concentrations along the root The LDH induction in the root axes, which are more resistant to long term anoxia than root tips, may indicate LDH contiues to energy mebolism in these more matre zones. This resh was supported, in part, by USDA grant 93-3710048922.

PROTEIN BINDING SITES IN THE PROMOTER OF A TOBACCO PHYTOALEXIN BIOSYNTHETIC GENE. JEFFREY D. NEWMAN & Joseph Chappell. Agronomy Dept., Univ. of Kentucky, Lexington, KY 40546-0091. When plants are challenged with pathogenic organisms or fungal elicitors, one response is the production of antimicrobial phytoalexins. Tobacco and other solanaceous plants synthesize sesquiterpene phytoalexins, which are derived from the isoprenoid biosynthetic pathway. The first unique step in the synthesis of the tobacco phytoalexin capsidiol is a cyclization of farnesyl pyrophosphate (FPP) catalyzed by 5-epi-aristolochene synthase (EAS), a sesquiterpene cyclase. Earlier work demonstrated the elicitor-inducibility of this enzyme activity, protein and mRNA, implicating transcriptional regulation of this gene. Examination of the EAS promoter for binding by proteins in nuclear and whole cell extracts revealed one very strong and several weak binding activities. In gel retardation assays, one DNA binding activity showed a high affinity interaction with a sequence at -240 (TAC box - TCTQAG]_T) and weaker interactions with similar sequences at -725 and -155. While the functional importance of the -240 TAC box is not yet known, preliminary transgenic tobacco data (see poster by Yin and Chappell) suggested that an elicitor-responsive cis-element is located elsewhere in the promoter. A binding activity that interacted wealdy with this utative regulatory region was identified and enriched by DNA affinity chromatography. The sequence to which this activity binds has been localized to a 27bp region that includes the tandem imperfect repeat AGACCCCAGACGCC. This sequence resembles both the AGC-box (AGCCGCC) associated with pathogenesis-related (PR) genes (Hart et al., Plant Mol. Biol. 21:121, 1993) and the lower strand of the chitinase elicitor-responsive element (EIRE) CTGACN3CTGAC (Fukuda & Shinshi, Plant Mol. Biol. 24:485, 1994). The effect of mutations in this region on DNA binding activity will also be reported. This work has been supportod by the National Science Foundation.

573

576

CONSENSUS SEQUENCES FOR PLANT MINIMAL PROMOTERS Sivashankary Yuwarai, ELIZABETH A. DAVIS Steven Ackerman Biology Department, University of Massachusetts, Boston MA 02125 An array of 278 minimal promoter sequences from plant genes was assembled from GenBank and analyzed. Sequences were aligned by designated TATA region or transcription start site. The consensus sequence for the TATA region was CTATAAA A. Sequences around the start site (Q) had a consensus of irICTcA_CcAA. The region between these two elements was AT rich, in contrast to the comparable region in animal promoters, which is GC rich. Thus, binding of TATA Binding Protein and other general transcription factors to minimal promoters may exhibit differences in plants and animals. This research was supported by the National Science Foundation grants DMB 8608207 and 9118667.

INDUCIBILITY OF VARIOUS HEXAMER MOTIF-CONTAINING UPSTREAM ELEMENTS IN NOPALINE SYNTHASE(NOS)PROMOTER YOUNGHEE KIM' ,Sunyoung Park', Jungyoon Park', Wonju Leel,Inshin Jeungl,gyoungok Kim' and Gynheung An2 'Dept.of Horticulture, Sangmyung women's university,Chonan,Korea; 2Dept. of Life Science, Pohang University of Science and Technology, Pohang,lKorea The nos gene located within the Transfer-DNA(T-DNA) of Agrobacter-iua Is readily expressed in plant tissue upon transfer to the plant chromosome. The nos promoter activity Is Inducible by mechanical wounding, salicylic acid and further enhanced by auxin. The nos promoter consists of the TATA box, and CAAT box, and upstream regulatory region. The 5' regulatory sequence located upstream of the CAAT box region is essential for the nos promoter activity. Deletion analysis of this upstream region showed that the region located between -130 and -112 is necessary for the nos promoter activity. A 20 nucleotide sequence containing two hexamer motifs and a spacer region between them is sufficient to restore the responsiveness of the minimal nos promoter(-101) to auxin, salicylic acid, and other stimuli.Other hexamer containing-elements,such as nos-RP,ocs and as-l,when substituted for the nos element, showed similar results for these stimuli in transgenic tobacco plants.

114

577

580

ARABIDOPSIS GBF3 IS REGULATED BY ABSCISIC ACID AND INTERACTED WITH G-BOX AND NON G-BOX ELEMENTS OF THE ADH PROMOTER GUIHUA LU & Robert 1. Ferl Program In Plant and Cell. Biol. Hort. Sci. Dept., Univ. Florida, Gainesville, FL 32611 The G-box motif in the Arabidopsis Adh promoter is one of the functional cis-acting element and is bound by a nuclear protein complex in vivo. Using an Adh G-box oligonucleotide screening approach, a full length cDNA clone, designated as GBF3, was isolated from an Arabidopsis suspension cell cDNA library. GBF3 encodes a 41-kDa bZIP protein that is homologous to the shortened root-specific Arabidopsis GBF3 (Schindler et al., 1992. EMBO J. 11: 1261-1273). Band shift competition assays combined with DMS and DNase I footprinting analyses showed that GBF3 expressed in E. coll sequence-specifically interacted with the Gbox (-210, 5'CCACGTGG3') and non G-box (-190, 5'CCAAGTGG3') elements of Arabidopsis Adh promoter. Although previous data indicate that the non G-box element may not interact with GBFlike factors, GBF3 did bind to this element in a sequence-specific manner in vitro. Deletion analysis indicated that the first 3 leucine repeats in GBF3 hexa-repeat leucine zipper domain were sufficient for its sequence-specific DNA binding. The transcription of both GBF3 and Adh genes was significantly increased by exogenous ABA in Arabidopsis suspension cells. These data suggest a potential signaling role of the interaction between cis-acting elements and GBF3 in the responsiveness of Arabidopsis Adh gene to environmental stresses, such as the induction of Adh by cold and dehydration (Dolferus., et al., 1994. Plant Physiol. 105: 1075-1087). This work was supported by NIH R01 GM40061 and USDA/NRI 93-37304-9608 to R. J. Ferl.

EFFECTS OF WINTER TEMPERATURES ON GROWTH AND APICAL DOMINANCE OF PINUS TAEDA WILLIAM C. CARLSON' & Constance A Harrington2 'Strategic Biology Res. Dept., Weyerhaeuser Co., Tacoma, WA 98477; 2Pacific Northwest Res. Stn., Olympia, WA 98512 Pinus taeda L. is generally considered to have a non-obligate chilling requirement with budbreak occurring more quickly if plants receive temperatures betwecn 0 'C and 8 'C. Two families from the North Carolina coastal plain were planted at three elevations (300 mn, 640 m, and 1065 m) on Maui, Hawaii. The two lower sites never had temperatures < 8 'C during the six years of the study; the highest elevation site experienced only a few hours < 8 'C each year. Lack of normal budbreak in the spring was conmmon at the lowest elevation site, particularly for one family. When normal budbreak did not occur, new terminal growth originated from buds of fascicular origin or those in non-terminal positions. Annual height growth at the two higher eleation sites was excellent (> 1 m yr'). Growvth ofthe tvo families was equal at the highest elevation site but dissimilar at the twvo losver elevation sites, thus indicating a genotype-by-environmcnt intcraction. Hcight growvth in the spring or summer was correlated with winter temperaturcs; number of hours per winter . 16 `C was positively correlated with subsequent growth (R > 0.80). The relationship between winter temperature and terminal grouth reversed sharply between 16 'C and 19 'C. The results suggest that for Pinus :aeda surprisingly warm winter temperatures will promote dormancy release and subsequent growth; howvever, some time period at colder temperatures (< 16 'C) is apparently required, at least with patterns of photoperiod present on Maui (latitude 20' N). The < 5 °C ineases in winter temperatures predicted by some climate change scenarios are unlik-ely to affect dormancy release in this species; howveser we would ecpect genotypic variation in groivth responses to elevated temperatures.

578 TRANSCRIPTIONAL REGULATION OF THE NUCLEUS-ENCODED CHLOROPLAST RIBOSOMAL PROTEIN GENE rpsl 7 IN ARABIDOPSIS 7HIAIANA JUI-SUI HSU & J en Gantt Department of Plant Biology, St. Paul, MN 55108 The synthesis of chloroplast ribosomes requires the expression of genes from both nuclear and chloroplast genomes in higher plants. In order to understand the coordinated expression of these genes, we started our investigation from the transcriptional regulation of the Arabidopsis thaliana nuclear gene rpsl7, which encodes one of the chloroplast ribosomal proteins. Nuclear extracts contain a DNA binding factor, Sl7F, that alters the mobility of the rpsl7 5'-flanking region in mobility-shift assays. By performing deletion and linker-scanning analyses, the target sequence of S17F was confined and revealed. The binding site is located about 45 bp upstream of the putative transcriptional start site. Because this is near a potential TATA sequence, we considered the possibility that the DNA binding activity was related to the TATA binding factor (TBF). To address this possibility, fragments of the CaMV 35S promoter were used to compete for S 17F binding in mobility shift assays. These experiments showed that while the 35S promoter does bind S17F, fragments containing the TATA sequence do not compete for its binding. The portion of the CaMV 35S promoter that contains the S17F binding activity was shown to be the region designated B3. Previous research by Benfrey and Chua [(1990) Science 250:959-966] has shown that transgenic tobacco seedlings containing a B3-TATA-uidA (encoding B-glucuronidase (GUS)) transgene express high levels of GUS activity only in green tissues. These data correlate well with our northern blot and GUS-reporter gene data and, together with CaMV 35S promoter-protein binding data, raise the possibility that S17F is related to CAF, the B3 binding factor.

581 THE EFFECT OF CHILLING ON THE RELEASE OF BUDS FROM ENDODORMANCY IN PICEA ABIES School of Natural BETTY JEAN MURRAY and Harrison Morton Resources and Environment, Univ. of Michigan, Ann Arbor, Ml 48109-1115 Regulation of bud growth by changes in the external environrmnt is extremly ccmplex. Our previous work suggests that the start of endodormancy (internally controlled within the bud) is concanitant with the cessation of onbryonic shoot growth in October. Endodormancy can be broken by an outdoor chilling period in Novenber, and a normal growth flush will follow under favorable greenhouse conditions. We now wanted to examine the conditions which fulfill chilling requirements and lead to growth. Forty 3-year-old Norway spruce transplants growing in Nichols Arboretun nursery, were dug on September 26, 1994, and potted in 5-gal pots. They were placed in natural light in a greenhouse or outdoors. On October 10, 18 plants fron the greenhouse, were moved to a growth chanber for chilling under SD (9.5 hs). Temperatures were 6VC (night) and 17'C (day). On Novenber 4, 3 more plants were added to the charber. On Novenber 10, the chamber environment was changed to one favorable for prarmtion of bud growth using 13 hs light. Temperatures were 12C (night) and 25C (day). More than 50% of buds on all plants receiving 31 to as few as 6 days of chilling, produced normal growth after 49 days fran start of preation. Buds indoors without chilling failed to resume growth as did those outdoors. It is chilling followed by a prcaotive condition that induces the resunption of growth. A research grant fran SNRE supported this work.

582 579 FACTORS ENHANCING THE EXPRESSION OF AUROISACJEKIUM VIRULENCE GENE AND TRANSFORMATION EFFICIENCY OF RICE Ming-Tsair Chan1, HSIN-HSIUNG CHANG2, Su-May Yu1 1 Institute of Mol. Biol., Acad. Sinica, Nankang, Taipei, TAIWAN 2 Dept. of Agronomy, Natl Taiwan Univ., Taipei, TAIWAN, ROC The activity of B-galactosidase could be used as a measurement for

de'ermining the capability of activiting Vi.Dgene in strain. The objectives of this study were to

AgkobacteA..um

characterize and to optimize the expression of transgenes in rice mediated by A. tume6acane . The phenolic compounds exudated from various rice tissues were assayed with HPLC. Results indicated that only 4-hydroxyacetophone (HAP) was present in the tissue exudates of eight rice varieties. Potato suspension cultures (PSC) and acetosyringone (AS), added in the infection medium, were found to be able to induce remarkably the expression of chimeric gene and enhance the transformation efficiency of mature rice embryos mediated

by Tiplasmid carrying mAmy B promoter/B-glucuronidase (GUS) Integration of foreign gene into the genome of transgenic tissue was confirmed by the inverse polymerase chain reaction (IPCR) analysis and expression of GUS was determined by Northern and Western blot analysis. gene.

DEVELOPMENTALLY ARRESTED MUTANTS IN ACETABULARIA: COMPLEMENTATION BY A WILD TYPE NUCLEUS OR WILD TYPE CYTOPLASM IN MUTANT:WILD TYPE CELL GRAFIS. Brenda Hunt & DINA F MANDOLI. Dept. Botany KB-15, Univ. Washington. Seattle. Washington 98195-1800 We have isolated 6 spontaneous mutant cell lines that are developmentally arested (das) in the vegetative portion of growth. These cells are "immortalized": they do not reproduce but are extremely long-lived compared to wild type. Some of mutants make sterile hairs (the whorls of sterile hairs that encircle the vegetativc stalk are characteristic of the phase of development) but most do not. Four appear dominant and 2 appear recessive. Based on terminal morphology, das-1,-2,-S and -6 arrest during juvenile growth and das-3, and -4 arrest dunng adult growth. das-5 and -6 tend to have branched stalks. das-2 segregated as a single, recessive Mendelian trait whereas das-S segregated as if it were dominant in the original self-roass from which these mutant lines were obtained. das-2 and -5 have whorless terminal phenotypes. Wben the mutant apex is removed. the nucleated portion of each mutant cell line repeats development and arrests again at with the same terminal phenotype attained prior to amputation. However, the enucleate cell body of dos-2 and das-5 can make a whorl implying that the mutant nucleus prvents full expression of the potential of the apex. In heterokaryotic grafts between das and wild type rhizoids (where the diploid nucleus resides), das-2 and das-5 were each rescued by replacing the mutant apex with a rhizoid of a wild type cell. Such heterokaryons produce from 24 caps most of which are fertile. Gametangia from thesc caps produce progeny of wild type and das when selfed. It is not yet clear whether there is a "1 nucleus: 1 cap' rule in such heterokaryotic grafts i.e. if the haploid products from the grafted nuclei are loaded onto distinct cytoskeletal tracks during nuclear transport. When the mutant apices of das-2 or das-5 are replaced by a wild type apex, the mutant phenotypes are complemented i.e. the grafted cells complete development and produce progeny. Phenotypes of the progeny from these grafts and epistasis relationships between das-2 and das-5 will be discussed. Supported by a grant from the National Science Foundation IBN-9305473 (DFM).

115

583 REPRODUCTIVE

ONSET IN ACETABULARIA

ACETABULUM: CHARACTERIZATION OF A DEVELOPMENTAL LOOP. Bend Fester Philip A, YoungandnDINA EMANDOUL University of Washington. Department of Botany, KB15, Seattle, Washington 98195. Organisms use intemal and external cues to time the entry into reproduction. Once the unicellular green alga Acetabularia acetabulum attains a certain phase of devdopment blue light tnggers the onset of reproduction, resulting in morphogenesis at the cdl apex to form a reproductive structure, or cap. Photobiological, cellular and molcular studies will indicate when and how this switch from vegetative to reproductive growth occurs and will allow us to study the attendant shape changes at the subceliular level. Cap initiation is optimal betwecn 19 and 210C and above 125 molnm2s-1 col white light. We define the first shape change of cap initiation distinguishable by gross morhoogy as knob (t=0), subsequent shape changes at the cell apex ar velum (t=lOh), inferior corona (t=25h), gametophores (t33h) and superior corona (t=55h). After knob, cap formation is independent of light. If a recently initiated cap is amputated, the cell will recapitulate developsnt. repeating a portion of vegetative morphogenesis (whorl formation) before re-inititating a new cap. Prcliminary data indicate that the developmental sequenc between amputation and reinitiation does require light: after two weeks. 96% of cdls amputated in lisht have re-initiated a second cap (n=50), while 0% of cells amputated in dark have re-instiated (n=50. Additionally, mutants isolated by inbreeding appear to be stuck in a developmental loop which includes cap initiation. These cells- abort reproduction repetitively, repeating a defined sequence of vegetative and reproductive growth. 71% of cells in strain MH have 1 or more abortions, as compared to 4-5% of cells in a wild-type population. The 3:1 ratio of aborting to non-aborting cells in strain MH suggests that the abortion phenotype, stuttercap, may be due to a dominant allde of a single gene. Rescue of these cells by amputation and cell grafting is currently underway. Supported by a Developmental Biology Training Grant from NIH (RF), an REU fellowship from NSF (PAY & DFM), and NSF grant IBN-9305473 (DFM).

584 INDOLE-3-ACETIC ACID TRANSPORT IN FUCUS GLORIA K. MUDAY D. Shari Ramcharanl. Ralph nuatrano2 Leigh Br*anX Amy ArnoldQ. 'Biology Department, Wake Forest University, Winston-Salem, NC 27109, 2Biology Department, University of North Carolina, Chapel Hill, NC 27599 Auxin transport has been implicated in controlling polar development in embryos of land plants. We have examined the developing zygotes of the brown alga, Fucus, in order to determine if auxin transport may play a role in the well characterized polar development of these zygotes. lTe presence of free indole-3-acetic acid (IAA) in both zygotes and mature tissues of Fucus was verified by gas chromatography-mass spectroscopy (GC-MS) with a total ion chromatograph. The levels of free IAA in the mature tissues were measured by GC-selected ion monitoring-MS and found to be 9.3 ng/gram fresh weight of tissue, with higher concentrations found in zygotic tissue. The IAA concentration in Fucus frond tissue is about half the concentration found in most land plants. The ability of zygotes to accumulate radiolabeled IAA was measured and this accumulation was shown to be sensitive to the auxin transport inhibitor, napthylphthalamic acid (NPA). NPA, which inhibits IAA efflux, increased the accumulation of IAA by up to 1.8 fold. This value is comparable to that found in tissues from land plants. The presence of NPA binding activity in microsomal membranes derived from zygotes was determined using filtration assays to measure the binding of [3HJNPA. From these analyses, the dissociation constant (Kd) for NPA was found to be 21 nM and the abundance of NPA binding activity (B,.,,) was 9.4 pmolelmg, values which are comparable to those from flowering plants. NPA binding activity partitions with the cytoskeletal elements during detergent solubilization, as has been seen previously with NPA binding activity from membranes of zucchini and maize. In conclusion, developing zygotes of Fucus possess endogenous indole-3-acetic acid and have the characteristics of a regulated auxin efflux carrier. (This research was supported, in part, by National Science Foundation grant IBN-9318250 to G.K.M and MCB-9318757 to R.S.Q.)

585 ISOLATION AND CHARACTERIZATION OF A -y-TUBULIN cDNA FROM THE MOSS PHYSCOMITRELLA PATENS TANYA A. WAGNERI. Jochen Schwuchow-. C. Elizabeth Oakley, Berl R.Oakley-. and Fred D. Sackl Depts. of 'Plant Biology and 2Molecular Genetics, The Ohio State University, Columbus, OH 43210 'y-tubulin is an important component of microtubule organizing centers in animal and fungal cells, but its role in plants is less well understood. We are cloning ry-tubulin from moss to investigate the possible roles of 'y tubulin and microtubules in gravitropism and tip-growth in moss protonemata. Degenerate primers to conserved regions of -y-tubulin were used to obtain PCR products from Physcomitrella genomic DNA. A 'y-tubulin PCR product was then cloned and used to screen an amplified Phlyscomitrella cDNA library, constructed in XgtlO. Of eight cDNAs isolated, the longest cDNA, X-y6, was cloned and one strand, 1,500 bp, has been sequenced to the poly A tail. This cDNA, which contains an open reading frame of 395 amino acids, is missing approximately 240 bp of coding sequence from the 5end, but shares 96% amino acid identity with a fern y-tubulin, and at least 90% identity with -tubulins from Arabidopsis and maize. Southern hybridizations suggest that there may be more than one -y-tubulin gene in Physcomitrella. Experiments are underway to obtain the 5' end of this -y-tubulin gene and to determine its expression level in moss protonemata. (This research is supported by a NASA GSRP fellowship NGT-51281)

116

586 A COMPARISON OF SPORE GERMINATION BETWEEN NEW JERSEY AND NOVA SCOTIA POPULATIONS OF SCHIZAEA HELEN GUIRAGOSSIAN KISS & Christy Byer Department of Botany, Miami University, Oxford, OH 45056 It is well known that red light promotes and blue light inhibits spore germination in many fern species. A previous study of the photoregulation of spore germination in the fern Schizaea pusilla from New Jersey demonstrated that maximum germination can be achieved if a dark and cold treatment preceded a two week red light treatment. Another study using spores from Nova Scotia reported that germination occurred in less than a week. In order to examine this major difference in the timing of germination events in Schizaea populations, we collected Schizaea spores in the fall of 1994 from two bogs in Nova Scotia and from one bog in New Jersey. We have found that Schizaea spores do not germinate in the dark in New Jersey and Nova Scotia populations. Dark and cold treatments enhance germination in both Nova Scotia and New Jersey populations. However, spores from New Jersey need greater than 10 days of continuous illumination for germination while Nova Scotia spores need 18 days or more. We are presently examining the effects of red, blue and far-red light on spore germination in both populations of Schizaea. 587 CONSTRUCTION OF A GENETIC MAP OF CARICA PAPA YA AND MAPPING AND GENETIC CHARACTERIZATION OF THE SEX) LOCUS THAT CONTROLS PLANT SEX. JOHN 1. STILES'. Suresh N. Sondur .2, James Deputv 'and Richard Manshardt t. Departments of Plant Molecular Physiology' and Horticulture2, College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI 96822. Randomly amplified polymorphic DNA (RAPD) technology has been utilized to construct a genetic map of papaya (C. papaya L.) using F2 plants from a cross between the Hawaiian variety 'Sunrise' and University of Hawaii breeding line 356. A total of 596 10 base synthetic DNA primers were screened against the parents and the F, and 96 polymorphisms were detected. At LOD 4.0, 62 of these markers mapped to 11 linkage groups comprising 999.3 cM. About 80% of the markers showed the expected Mendelian segregation ratios. The single locus that controls plant sex, SEX), was mapped to a 14 cM region with flanking markers 7 cM on each side. In papaya, female plants contain a recessive allele at SEXI and have the genetic composition sexl-flsexl-f Hermaphrodite or male plants are heterozygous for a dominant hermaphrodite or male allele of SEXI (SEXI-H or SEXI-M). Homozygous dominant allele-containing plants are not viable. Recombination in the vicinity of SEX) is distorted. Recombination is suppressed in hermaphrodite plants (SEXI-Hlsexl-f). Recombination events between SEX] and the flanking markers occun-ed 10 times more frequently in female plants than in hermaphrodite plants. There may be a chromosomal aberration associated with the SEXI-Hand SEX)-Malleles. Additional RAPD markers linked to SEX) have been obtained by bulked-segregant analysis. We are now doing physical mapping in this region using cloned RAPD fragments as hybridization probes.

588 A FAMILY OF NOVEL CYCLIN SEQUENCES EXPRESSED DURING DIRECT

SOMATIC EMBRYOGENESIS IN ALFALFA Fugenia =,usina Adrian Slael, Atanas 1. Atanasgov and MALCOLM C. Ft I110T; Norman Borlaug Inst. Plant Sci. Rcs.. De Montfort Univ. Leicester LE7 9SU, UK A procedure has been developed for direct production of somatic embryos from alfalfa leaf explants. The leaf explant is wounded then subjected to 2.4-D treatment which induces competent cells in the leaf to undergo the asymmetric division which is the first visible stage of commitment to embryo formation. It will be necessary to understand the regulation of this asymmetric division is order to elucidate the nature of embryogenic competence and determination. Gene sequences known to be involved in the control of cell division have been isolated from tissues in which competent cells are progressing through the early stages of direct somatic embryogenesis. A cdc2 fragment isolated by RT-PCR amplification using degenerate primers is identical to the alfalfa cdc2MsA gene. However, a cyclin sequence amplified using the same approach differs markedly from the mitotic cyclins which have previously been identified in alfalfa. The sequence of this PCR product was used to design primers for 5- and 3-RACE procedures. Analysis of 5-RACEproducts revealed considerable sequence heterogeneity in the N-terminal region of the coding sequence. with at least two cyclin sub-families apparent. The complete coding sequence of one member of this novel cyclin family has been obtained by the RACE strategy. The role of this novel family of cyclins in the first embryogenic cell division is currently under intensive investigation.

589 THE EFFECT OF ACTIVATORS AND INHIBITOR OF PROTEIN KINASE C ON SOMATIC EMBRYOGENESIS OF RAPID CYCLING Brassica rapa WOO Y. YANG. Zeung K. Cho & Timothy J. Mulkey, Life Sciences Dept. Indiana State University Terre Haute IN 47809 Rapid cycling brassicas are being used to investigate many problems in plant biology and as tools in education. Brassica rapa immature seeds of different cultivar .vere tested for the ability to initiate embryogenic cultures. Nonembryogenic callus vas induced in several cultivars of B. rapa cultured on media supplemented with various concentration of 2,4-D. But embryogenic callus wvas obtained ivhen B. rapa cv. rar wmas cultured for 4 weeks on MS medium containing various concentration of 2,4-D and then transferred to the same medium containing 5 mg/I of 2,4-D. After I wveek of culture of embryogenic callus of B. rapa cv. rar on MS hormone-free liquid medium, somliatic embryos present a functional bipolar organization with a fused cotyledon and a comiipletely differenitiated radicle. Treatmeint of I ss'eek old sonlatic embryo with protein kinase C activating agents (TPA and DAG) increased in multiple shoot development fromil cotyledon region of somatic embrytos. Concentrations of 107 aid 10 ' M mTPA Yhicil is knowis as inhibitor of protein kiniase C increased adventitious root developmenlt. These results suggest that the activity of protein kinases niny be part of the action regulated by hormones during bipolar developimient of sonstic embryo. Using the protein phosphorylationi analysis, we are determining the effeet of PKC on in vitro plant regeneration.

590 BIOCHEMICAL CHANGES OF POLLEN DURING HYDRATION, GERMINATION, AND TUBE GROWTH: MEASUREMENT OF pH AND APPLICATION OF FrIR J. Henr Sionel, MaIry Eve McCutchenl, & LeRoy Peterson Jr. 2 1Dept. of Biol. and 2Dept. of Chem. and Physics, Francis Marion University, Florence, SC 29501 Using conventional methods and Fourier Tmnsform Infrared Spectroscopy (FTIR), we have begun an investigation of the biochemical and biophysical changes that occur during the hydration, germination, and tube growth phases of pollen from Impahieris sultanii. Germination and growth rates of pollen tubes incubated in a weakily buffered (1 mM Mes) medium were compared to that incubated in an unbuffered medium. Germination and tube growth were rapid in the medium buffered at a starting pH of 6.80. During the tube growth phase, the pH in this medium remained constant at 6.75 after dropping to that value during the preceding 10 minute hydration and germination phase. In the unbuffered medium, adjusted to a same starting pH, tube growvth was almost as rapid despite the fact that the pH fell precipitously to 5.9 during the hydration and germination phase and remained at that value during tube growth. In contrast, when pH values were less than 5.3 in either media during the growth phase, tube growth was severely inhibited. Thus these pollen tubes do not acidify the medium surrounding them during the growth phase and do not require substantially acid conditions to grow rapidly. Noninvasive probing using FIR indicated numerous biochemical and biophysical changes, including changes in the intensity and contour of infmred bands as well as shifts in band frequency during the entirc incubation period. Preliminary results and tentativ-e assignments of band frequencies indicate major changes in CO2 concentration (absorbance intensities at 2327 and 2355 cml); changes in the secondary structures of proteins (shifts in amide I and II frequencies in the 1660 to 1630 cm- 1 and 1550 to 1540 cm- 1 regions); changes in amino acid side groups of proteins or changes in acyl chain packing or conformation of lipids (a moderate shift in frequency of CH2 or CH3 bending vibrations from 1450 to 1440 cm-I); and a complex pattern of major changes in phospholipid head groups andlor carbohydrates (intensity changes and frequency shifts in 950 to 1100 cm- 1 region).

592 ISOLATION OF THE ARABIDOPSIS HOMOLOGS OF TWO GENES EXPRESSED IN PHALAENOPSIS OVULES: A HOMEOBOX GENE AND A GENE OF UNKNOWN FUNCTION Jeanette A. Nadeau. Elena R , Alan J. Williams and SHARMAN D. O'NEILL. Section of Plant Biology, Division of Biological Sciences, University of California at Davis, Davis, CA 95616 The development of the angiosperm ovule has been well characterized descriptively, but to date there is little information about the molecular mechanisms which lead to the differentiation of the specialized tissues of the ovule. Our laboratory has begun the molecular characterization of ovule development by isolating cDNAs from the developing ovules of Plialaertopsis orchids by differential screening. These cDNAs represent transcripts that are expressed only in ovule tissue, and which exhibit stage-specific expression patterns suggesting a role In pivotal events during ovule development. To further our understanding of the function of these genes, we have isolated the homologous cDNAs from Arabidopsis, a species in which genetic and transgenic studies will be possible. Here we present information about two classes of genes isolated from Arabidopsis that are of particular interest; a homeobox gene (A20) and a gene of unknown function which is expressed only in mature, receptive ovules (A108). Homeobox proteins bind DNA in a sequence -specific manner so as to regulate the expression of target genes during development. Recently, a number of homeobox genes have been Isolated from plants, including the Knottedl-like genes which appear to play a role in maintaining the indeterminacy of plant cells. Sequence analysis of A20 suggests that it falls into a new family of plant homeobox proteins that may play a role in cellular specification events, which includes the Glabra2 gene which is necessary for trichome differentiation. Sequence analysis of A108 shows that it is a member of a family of related sequences in Arabidopsis, with no known function. We will describe the temporal and spatial expression patterns of these two genes in Arabidopsis and relate this to the patterns observed in the monocot species Phalaenopsis. This research was supported by a grant from USDA/NRICGP to S.ON.

593 CHARACTERIZATION OF LIPOXYGENASE EXPRESSION IN RIPENING TOMATO FRUIT KURT D. KAUSCH and Avtar K. Handa. Horticulture Department, Purdue University, West Lafayette,IN 47907-1165. We have cloned a lipoxygenase(LOX) gene encoding an enzyme that accumulates to maximal levels at the red-ripe(RR) stage of tomato (Lycopersicon esculentum Mill) fruit development. The complete nucleotide sequence of the LOX cDNA (2871bp; GenBank accession number U13681) was determined and is representative of other reported plant LOX sequences (between 60 to 100% homology). Polyclonal antibodies were raised to LOX protein purified from RR tomato fruits and used to determine spatial and temporal distribution and accumulation of LOX during fruit development using immunocytolocalization. Results revealed an instial appearance of LOX in the locular jelly from mature green fruit followed by a gradual accumulation in the columella, radial wall and pericarp regions during the course of fruit ripening. Ripe fruit had the greatest accumulation of LOX protein in the locular jelly followed by pericarp and placenta tissues, respectively. Quantification of LOX protein in extracts obtained from different fruit tissues by SDSPAGE and LOX antibodies showed minimal accumulation in all unripe tissues compared to an abundance of LOX protein in all ripe tissues especially the radial wall, locular jelly and endopericarp regions. Detectable LOX activity was found only in exo- and endopericarp tissues of mature green fruit and in all ripe fruit tissues except placenta and locular jelly. The basis of this apparent discrepency, i.e. a large accumulation of LOX protein but little enzymatic activity in the locular jelly, is under investigation. Steady state levels of LOX mRNA in different tissues of unripe and ripe fruits are being determined to further localize expression of the cloned LOX gene. This research was supported by USDA/CPBR grant 90-34190-5207.

591

594

MOLECULAR CLONiNG OFAN ANTHER SPEOFIC GENE FROM TOMATO

NATIURI ANI) ROt)ti ItFNIIANCIED) RLSl'tRAItON DUIRIN(i SP'ROItrING OF AGED PO0TATO SEEI.-TUBERS. Cl. N. MOIt[AN KUMAR said N. Richard Kix'wtcs, Deparunetit of Agricultural. Food and Nutritional Scienice, Univ. of Alherta, Edimiontion, Alberta, Canada T6G 2P5 Aging during long-tenn storage (up to 30 months, 40C, 95% R.lu.) of potato seed-tubers results in increased respiration during sprouting and a loss of sprout vigor. To characterize the nature of the age-enhanced respiration, sub-mitochondrial particles (SMP) were isolated from young (7-mo-old) and old (19-mo-old) ned-tubers at 0 (directly from 4°C storage). 5, 10 and 20 d of sprouting. SMP proteins were compared on SDS-PAGE and were probed for altemate oxidase and ATP synthetase (OSCP & psubunits) by immunioblotting. Alternate oxidase was not present in intact seed-tubers of either age; however, comparison of trends in adenylate concentrations and energy charge, along with greater amounits of ATP synthetase in older tubers during sprouting, indicated that respiration was fully coupled and cyt-mediated. IHigher respiration of sprouting older tubers was likely a response to a lower AEC prior to sproutiuig, since inicreased respirationt durinig sprouting rcsulted in raising the AEC to equal that of younger tubers. The reduced sprout vigor and higher ATP production of older tubers suggest that older tubers must expend more metabolic energy in processes which arc not directly liinked to sprout growth. In view of the increased free-radical (FR)-mediated peroxidative damage associated with tuber aging (Plant Physiol. 102:115-124), we examined whether glutathione (GSH)-mediated FR-scavenging was a greater sink for ATP/NADPH in sprouting older tubers. Older tubers had higher concentrations of reduced (GSH) and oxidized (GSSG) glutathione, and higher glutathione reductase and trnsferane activities than younger tubers prior to, and during sprouting. The reduction of GSSG to GSH is NADPH dependent, while GSH synthesis requires ATP and glutamate. Thus, GSH-mediated FR-scavenging likely contributes to a lower AEC in older tubers prior to sprouting, which in turn effecs increased respiration during sprouting. This research was supparted. in part, by a grant from the Natural Sciences and Engineering Research Council of Canada.

C. DANIEL RIGGS and Andrea Horsch. Botany Department, University of Toronto, Scarborough College, 1265 Military Trail, Scarborough, Ontario MlC 1A4 Canada The primary focus of this laboratory is the identification and characterization of proteins and genes which regulate melotic metabolism. For these studies we exploit the natural synchrony of Lillum longiflorum meiocytes. To develop a tool for such investigations we generated a polyclonal antiserum directed against meotic prophase nuclear proteins which bind to DNA-cellulose. Immunoblotting of total cellular proteins from both meiotic and vegetative tissues revealed that the antiserum recognizes a number of proteins, and the onset and duration of expression of putative melosis-specific proteins was documented. A protein which first appears at pachytene was purified and N-terminal sequence analysis indicated that it was the product of a recently described cDNA known as UM9, which may be a serine protease. In order to conduct molecular experiments, we wanted to isolate a tomato cognate and use it in gene fusion experiments. We screened a tomato genomic library with the UM9 insert and report here the characterization of one clone which exhibits sequence similarity to LIM9. Preliminary hybridization results indicate that the tomato clone is expressed only in young anthers, suggesting that it perhaps encodes a melosis-specific gene. Recent prgress will be reported. Supported by NSERC grant #OGP0090057.

117

595 CYTOKININS INFLUENCE FLOWER DEVELOPMENT IN THE WILDTYPE AND HOMEOTIC FLORAL MUTANTS OF ARABIDOPSIS. S.P.Venglat. & V.K.SAWHNEY Dept. of Biology, Univ. of Saskatchewan, Saskatoon, SK, S7N OWO Cytokinin, benzylaminopurine (BAP), and a phenylurea derivative-thidiazuron (TDZ) with cytokinin-like activity, were applied on wild type and some floral mutants of Arabidopsis at three developmental stages; l.vegetative, 2.post-inflorescence initiation and 3.mature bud stage. At 10-4M, TDZ primarily affected stages 2 and 3. TDZ; A)induced vegetative characteristics in floral organs, i.e., branched trichomes on sepals and carpels, and stomata on petals, B)increased floral organ size, and C) induced the formation of 20 floral buds in the axils of sepals. BAP at 10-3M affected all three stages. The changes included; A)increase in floral organ number (as in clvl mutant), B)formation of abnormal floral organs i.e., sepal-petal and petalstamen intermediates, and C)the production of 20 floral buds in the axils of sepals (as in apl-1 mutant). BAP also induced 20 floral buds in the axils of perianth parts of ap2-6, ap3-1 and ag flowers. BAP-treated ap2-1 flowers produced carpel-like structures instead of is which ovules, characteristic of the strong allele, ap2-6. Experiments with TDZ and BAP suggest that cytokinins are likely involved in the control of floral meristem identity, and that they influence the later stages of differentiation in floral organs. This research was supported by the NSERC of Canada.

596 EXPRESSION OF MULTIPLE CELLULASE GENES DURING ABSCISSION OF TOMATO FLOWERS: RELATIONSHIP TO PEDICEL BREAKSTRENGTH EL delCAMILLQ1 and,Alan Bennet2. 1Dept of Botany, University of Maryland, College Park, MD 20742, 2Deparnent of Vegetable Crops, University of California at Davis, Davis,CA 95616 Tomato flowers abscise naturally if pollination fails. Comparison of breakatrength (BKS) distributions in air and ethylene demonstated Ihat prior to sbedding flowers become weaker but are not yet ready to separate. Abscising flowers pass through a series of different abscission stages empirically defined from BKS distributions. The transition between stages was then correlated with cellulase gene expression. RT-PCR with short degenerste

primers derived frt conserved sequences of plant culases and oal RNA frm flower abscission zone explants treated with C2H4 identified six different cellulase gene fragments. Four of the six genes are homologous to fruit pericarp aclulases and the other two represent new cellulase genes, referred to as CELs and CEL6. Expression studies in naturally abscising flowers and flower explants induced to abscise in air or C2H4 demonstrated that both new cellulases are correlated with flower shedding. The CELs message level increases during shedding but not during the early stages of weakening, and expression was confined to the abscission zone. In contrast, the CEL6 message level increases during weakening and then decreases during shedding. Interestingly, expression of CEL6 was high in stems and remained high after treatment of the stem with C2H4. Thus, the up regulation of CELs and down regulation of CEL6 is specific to the abscission zone. These results suggest that flower bscission may develop as a multi-step process involving several type of cellulase genes which are up and down regulated during the process. Southern analyses have demonstrated that CELs is a gene family with at least three members while CEL6 is a single copy gene and is conserved between tomato and Arabidopsis. Supported by a USDAINRI grant to EdC.

597 LONG RHIZOID MUTANT (LOR) IN ACETABULARIA: PHENOCONVERSION OF WILD TYPE RHIZOIDS BY LOR RHIZOIDS IN HETEROKARYOTIC MUTANT:WILDTYPE CELL GRAFT7S. JENNIFER NIGGEMEYER & Dina F Mandoli. Dept of Botany KB-15, Univ. WA, Seattle, Washington 98195 The ease of nuclear manipulations (e.g. transformation, karyotyping. staining. and nuclear implantations) in Acerabularia actabulum are complicated by physical inaccessiblity of the diploid nucleus. The rhizoids. which attaches the alga to its substrate and houses the nucleus, are short and fist-like in wild type cells. A spontaneous mutant phenotype in an inbred cdl line of Acetabularia acetabulum produces rhizoids (lor) which are long and spread out, making it easier to physically manipulate and observe the nucleus. lor cells have an average of 4.9 0.2 rhizoids which are 4-5 fold longer than wild type. A n intermediate phenotype, lor/2, makes both short and long rhizoids. Four out of 9 self crosse of different lor cells segregate 1:2:1 which might suggest that bor involves two loci nd is co-dominant except that the orginal cells were lor. One of the 9 self crosse prduced too few progeny to analyse (