▼ Stomata are epidermal pores surrounded by two guard cells that regulate gas exchange between the shoot and the atmosphere. Stomata form and are spaced through asymmetric divisions controlled by TOO MANY MOUTHS (TMM) and STOMATAL DENSITY AND DISTRIBUTION1 (SDD1). The function of these genes have been characterized during leaf development where both restrict divisions and stomatal development. Unlike SDD1, TMM, a likely receptor, has different functions in different organs e.g. excess stomata form in tmm leaves but tmm stems lack stomata. In tmm stems, initial asymmetric divisions were normal, and the resulting precursor cells, meristemoids, appropriately expressed cell fate markers. However, instead of forming stomata, meristemoids arrested, dedifferentiated, and elongated. Thus asymmetric divisions are necessary but not sufficient for stem stomata and TMM is essential for the transition of a meristemoid into a guard mother cell. SDD1, a serine protease, also regulates asymmetric divisions. To study their genetic relationship, tmm-1 sdd1-2 double mutants were analyzed. Double mutant leaves had a complex phenotype, partly resembling each single mutant and partly exhibiting novel phenotypes. This suggests that these genes act either in separate genetic pathways or in ones that converge on division regulation. However, double mutant stems lack stomata and thus tmm is epistatic to sdd1-2. This suggests that TMM is downstream of SDD1 in the same genetic pathway in this organ. SDD1 overexpression reduces stomatal number and TMM is required for this phenotype, a result consistent with an epistatic relationship. TMM is also required in leaves for SDD1 overexpression phenotype suggesting that this gain-of-function phenotype results from SDD1 misexpression. In a wild type background the overexpression of TMM showed no abnormal phenotype. Finally, SDD1 regulates the size of the stomatal stem cell compartment marked by TMM expression. Thus nature of the interaction between SDD1 and TMM depends upon whether TMM acts as a positive regulator of precursor cell development or as a negative regulator of asymmetric divisions with the phenotype depending upon the organ. Advisors/Committee Members: Lamb, Rebecca S.

Advisors/Committee Members: Transeau, E. N.

Advisors/Committee Members: Fisher, T. Richard.

▼ Transcription factors are emerging as key players in the origin of evolutionary novelties and as powerful tools to manipulating plant metabolism. The number of R2R3 Myb genes have amplified dramatically in the plants, where they are involved in the regulation of plant form and metabolic diversity. However, the function of most plant R2R3 Myb genes remains to be determined, primarily due to the lack of phenotypes of loss-of-function mutants. Here, we have investigated the evolutionary origins of R2R3 Myb genes and started to establish the functions of recently duplicated R2R3 Myb genes in maize through a gain-of-function approach. We have identified discrete evolutionary steps involved in the formation of plant specific R2R3 Myb genes from widely distribute R1R2R3 Myb genes. We have used the Black Mexican Sweet (BMS) maize cell culture system to investigate the consequences on the accumulation of metabolites of expressing the novel R2R3 MYB transcription factor ZmMYB-IF35. We demonstrate that, despite the high amino acid identity in the MYB domain with the P1 regulator of 3-deoxy-flavonoid biosynthesis, ZmMyb-IF35 does not induce the accumulation of flavonoids. However, similar to P1, ZmMyb-IF35 induces the accumulation of ferulic and chlorogenic acids, as well as several other compounds not found in the control BMS lines or in P1-expressing lines, suggesting a promising role of ZmMyb-IF35 for engineering the accumulation of various phenolic compounds. Together, our studies indicate that despite their recent common evolutionary origins, ZmMyb-IF35 and P1 have functionally diverged. Theses studies also suggest a possible correlation between phylogeny and function. Whether this is a general characteristic common to all regulatory genes remains to be determined. Advisors/Committee Members: Grotewold, Erich.

Advisors/Committee Members: Transeau, Edgar Nelson.

▼ In eukaryotes, the mitotic spindle assembly checkpoint (SAC) ensures the fidelity of chromosome segregation. The SAC acts through monitoring the attachment of microtubules (MTs) to chromosomes at the kinetochores. When not all chromosomes are attached with MTs in a bi-polar manner, components of the SAC on the kinetochores constitutively initiate signals to delay sister chromatid separation. Recently, it has been shown that the SAC components Mitotic Arrest Deficient 1 and 2 (MAD1 and MAD2) associate with the nuclear pore complex (NPC) during interphase and require certain nucleoporins, such as Tpr in animal cells, to properly localize to kinetochores during mitosis. In plants, although some of the SAC components including MAD2, BUR1, BUB3 and Mps1 have been isolated, the roles of SAC proteins during interphase and mitosis are still poorly understood and it is unknown whether they require plant nucleoporins for proper localization. In this study, we characterized the Arabidopsis homologs of human Mad1 and Mad2 (hMad1 and hMad2), AtMAD1 and AtMAD2, and their interactions with the Arabidopsis homolog of Tpr, NUA. An AtMAD2 T-DNA insertion mutant with lower expression of mRNA transcripts exhibits a shorter primary root and a smaller root meristem, very similar to a NUA null allele. Two other AtMAD2 T-DNA insertion lines with higher levels of mRNA transcripts exhibit a longer primary root, and an extended root meristem. AtMAD1 and AtMAD2 are localized at the nuclear envelope (NE) in interphase Arabidopsis root cells and their NE localization requires the plant nucleoporin NUA. This requirement for NUA appears specific, since depletion of another nucleoporin (Nup160) does not affect the localization of AtMAD2. AtMAD2 localizes to kinetochores during prophase and prometaphase and disassociates from kinetochores as the cell cycle proceeds in live BY-2 cells. Yeast two-hybrid assays demonstrate binding of AtMAD1 to AtMAD2 and NUA, as well as AtMAD1 dimerization. No direct interaction between AtMAD2 and NUA was detected. The interactions of AtMAD1 and AtMAD2, as well as between AtMAD1 and NUA were confirmed by co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC), respectively. Together, these data suggest that, in Arabidopsis, NUA scaffolds AtMAD1 and AtMAD2 at the nuclear envelope to form a functional complex and that both NUA and MAD2 are required for root development in Arabidopsis. Advisors/Committee Members: Meier, Iris.

▼ Sexual reproductive success may affect the long-term persistence of rare, clonal species. To determine if attributes of sexual reproduction in Trifolium stoloniferum contribute to the rarity of this federally endangered plant, I characterized its breeding system and natural levels of sexual reproduction in small and large populations. Inflorescences bagged at the bud stage failed to produce seeds, while hand-tripped (selfed) inflorescences set viable seeds, indicating that Trifolium stoloniferum is self-compatible but does not autonomously self-pollinate. Selfed seeds germinated well and developed into vigorous plants that did not appear to exhibit inbreeding depression. Senescing inflorescences were collected from two small (< 270 ramets) and two large (> 1000 ramets) southern Ohio populations in 1996 and 1997. I determined percent fruit set, the number of initiated seeds, and overall seed quality for each sampled head to measure sexual reproduction and test for differences among small and large populations. When data were averaged across years, percent fruit set and seed initiation per inflorescence were significantly greatest in the smallest population (mean = 73.3% and 61.6 seeds, respectively) and lowest in the largest population (11.0% and 6.3 seeds). Rainfall was abnormally high during the spring of 1996, and many of the initiated seeds failed to mature normally. Seed quality was associated with population in both years. The smallest population occurred at the most open site and consistently produced the highest quality seeds, while the largest population, located in a wooded environment, had the lowest seed viability. Since neither seed production nor seed quality was positively correlated with population size, even small populations may be worthy of preservation. My results do not suggest that overall seed production is the primary determinant of rarity, although dependence on pollinator service, low seed quality in unfavorable years, and low seed initiation and viability in the largest Ohio population may contribute to the endangered status of T. stoloniferum. These factors that potentially limit reproductive success may be more closely linked to habitat characteristics than to population size. Advisors/Committee Members: Snow, Allison A.

Advisors/Committee Members: Taft, Clarence E.

▼ F-box protein-mediated proteolysis is crucial for the circadian clock progression. ZEITLUPE (ZTL) is the only F-box protein so far identified in plants that regulates the circadian clock function. Little is known about the mechanism by which it is involved in the regulation of the circadian clock. The purpose of this study has been to investigate how ZTL activity and expression is regulated, what the functions of the ZTL domains are, and what are the factors that genetically interact with ZTL to maintain the proper function of the circadian clock. In this study, we first characterized an Arabidopsis suspension culture that contains a fully functional clock. Then, we used this cell culture to analyze the expression of ZTL. We found that ZTL expression is regulated at posttranscriptional step and the degradation of ZTL is circadian phase-specific. The phase-specific degradation of an F-box protein provides an additional level of feedback regulation in the interlocked feedback regulatory loops of the circadian clock. The LOV domain of ZTL is believed to function in light sensing. Recent in vitro data also indicate that LOV domains from other proteins are able to self-dimerize. In this study, we provide additional evidences to show that LOV domain of ZTL can interact with full length ZTL in vivo. High levels of LOV expression in Arabidopsis plants result in dramatic reduction of endogenous ZTL protein likely accounting for the longer periods phenotype of the transgenic plants. We also show that LOV interact with FKF1 another ZTL family member that acts in the clock-controlled long day flowering pathway to promote flowering. Therefore, high levels of LOV is likely to inhibit FKF1 activity or to promote FKF1 protein degradation, leading to late flowering phenotype of the plants that overexpress LOV. From a genetic screen for mutants that enhance or suppress the ztl-1 long-period phenotype, we recovered seven ztl-1 enhancer lines. The initial mapping effort locates one of the enhancer genes to the right arm of chromosome 4. The cloning of these genes would identify factors that genetically interact with ZTL in the regulation of the circadian clock. Advisors/Committee Members: Somers, David.

Advisors/Committee Members: Laurie, Alex.

Advisors/Committee Members: Transeau, Edgar Nelson.

Advisors/Committee Members: Transeau, Edgar Nelson.

Advisors/Committee Members: Stuckey, Ronald L.

▼ Not much is known about the efficient trafficking of potentially toxic phytochemicals from their site of synthesis to their correct intracellular destinations. Anthocyanins, the colored products of the well characterized flavonoid pathway, were used as a convenient model system to understand the cellular, biochemical and molecular processes involved in its transport from the cytoplasmic face of the ER to the central vacuole. Cell biological observations of maize Black Mexican Sweet (BMS) cells in culture, constitutively expressing the MYB (C1) and bHLH (R) transcription factors, show these cells to accumulate anthocyanins in multiple vacuoles with anthocyanic vacuolar inclusions (AVIs). Exposure to light caused calli to darken. This was attributed to the fusion of the vacuoles and ‘spread’ of anthocyanins from the AVIs into the sap but not due to changes in transcripts of the enzymes or the accumulation of pigments. Formation of vacuoles from dynamic pigmented compartments was observed in maize tassel glumes. These observations indicated an alternate vesicular route of transport of anthocyanins to the central vacuole. In Arabidopsis, I exploited the transparent testa mutant tt5, deficient in the enzyme chalcone isomerase (CHI) and its anthocyanin complementation with naringenin to develop an easily manipulatable seedling model system. Naringenin-treated tt5 and wt seedlings accumulated a new anthocyanin peak identified as cyanidin 3-glucoside. Global transcriptome changes were monitored using microarrays to identify potential transferases and transporters involved in either the detoxification of naringenin or the transport of anthocyanins. The induction of signaling components, jasmonic acid biosynthetic genes and defense-related stress response genes, suggested additional roles of flavonoids in several cellular processes. Lastly, the need for the catalytic function of CHI was investigated in driving flux into the flavonoid pathway. The catalytic mutants Y104F, T46A and R34A were generated in maize CHI (ZmCHI). In vitro assays demonstrated ZmCHIY104F to retain 20% of the ZmCHIwt activity while ZmCHIT46A and ZmCHIR34A were catalytically inactive. Only ZmCHIY104F and, surprisingly, the in vitro catalytically inactive ZmCHIT46A complemented Arabidopsistt5 mutants. These findings revealed additional roles of CHI in the flavonoid pathway. Taken together, these observations and results provided significant insights in understanding processes involved in phytochemical trafficking. Advisors/Committee Members: Grotewold, Erich.

▼ Long alpha-helical coiled-coil proteins are involved in a variety of organizational and regulatory processes in eukaryotic cells. In contrast to yeast and animals, only few such proteins have been investigated in plants. Here, two plant long coiled-coil proteins were investigated in detail, MAR-binding filament-like protein1 (MFP1) and the putative Arabidopsis homolog of Tpr (translocated promoter region). MFP1 is a nuclear-encoded, long coiled-coil protein that is targeted to plastids. It accumulates to comparable levels in all tissues of Arabidopsis which contain green chloroplasts, regardless of age and organ identity, but is much less abundant in roots of both light-grown and dark-grown seedlings. MFP1 protein accumulation parallels chloroplast development during the greening of seedlings shifted from dark to light, suggesting that MFP1 expression is regulated in a tissue-specific and light-dependent manner. MFP1 is localized in chloroplasts both in suspension culture cells and in leaves, and it is associated with the stromal side of thylakoid membranes of mature chloroplasts. It is co-purified with nucleoids, suggesting a function at the interface of the chloroplast genome and the photosynthetic membranes. MFP1 comprises a major DNA-binding activity in Arabidopsis chloroplasts and binds to several regions of the chloroplast DNA with equal affinity. Several thylakoid proteins are phosphorylated by a protein kinase CKII-like activity, and the alpha subunit of a chloroplast-located CKII has recently been identified as a component of the chloroplast transcription complex. Chloroplast-localized MFP1 is phosphorylated in vivo, and in vitro by CKII and phosphorylation inhibits its DNA-binding activity. A tandem CKII site in the DNA-binding domain of MFP1 was identified which is involved in the phosphorylation-dependent loss of DNA-binding activity. These features of MFP1 make CKII-dependent phosphorylation a possible mode of regulating the DNA-binding activity of the protein in vivo. Together, these data suggest that MFP1 is an interesting candidate for an architectural protein, possibly involved in anchoring nucleoids to thylakoid membranes. Tpr is a long coiled-coil protein associated with the nuclear surface of the nuclear pore in animals and yeast, where it is involved in mRNA export. A putative Arabidopsis Tpr homolog was identified. A T-DNA insertion mutant in its gene has a pleiotropic phenotype including early flowering, reduced apical dominance and morphological alterations of leaves and inflorescences. The connection between the predicted function of Tpr and its mutant phenotype is currently being investigated. In animals and yeast, the small GTPase Ran is involved in nucleocytoplasmic transport, spindle formation, and nuclear envelope formation. These functions are controlled by a RanGTPase-activating protein (RanGAP) and a guanine nucleotide exchange factor (RCC1). Vertebrate RanGAP1 is conjugated with the ubiquitin-like protein SUMO. SUMOylation of RanGAP1 is required for nuclear envelope-association in interphase and for spindle and centromere association in mitosis. Plant RanGAP lacks the SUMOylated C-terminal domain of vertebrate RanGAP, but contains instead a plant-specific N-terminal WPP domain, which is necessary and sufficient for targeting the protein to the nuclear rim. By examining the localization of Arabidopsis RanGAP1 during the cell cycle in stably transformed tobacco BY-2 cells expressing AtRanGAP1-GFP, we found that AtRanGAP1 localizes to the nuclear rim during interphase and to the cell plate during cytokinesis. A WPP domain-GFP fusion behaves like full-length AtRanGAP1-GFP, while WPP-domain deletion abolishes all targeting, demonstrating that the WPP domain is necessary and sufficient for both targeting events. Point mutations in conserved residues of the WPP domain abolish targeting to the nuclear rim and the cell plate, suggesting that the same mechanism is involved in anchoring RanGAP1 in both locations. These results imply a novel function of AtRanGAP1 during cell cycle and suggest a role of the Ran cycle in controlling cell plate formation in plant cytokinesis. Advisors/Committee Members: Meier, Iris.

▼ Plants encounter many different pathogens during their lifetimes, and they defend themselves through a combination of preformed and induced responses. Induced defense responses include basal defense, gene-for-gene resistance, and nonhost resistance activated on perception of non-self or the activities of non-self. To counteract plant defenses, plant pathogens possess weapons to avoid or suppress plant defenses. I investigated the molecular interactions between plant-pathogenic bacteria by using two model organisms, Arabidopsis thaliana and Pseudomonas syringae, and report here three discoveries. First, PAMP (pathogen-associated molecular pattern)-induced basal defense and R-protein mediated gene-for-gene resistance are linked (Chapter 2). I found that two P. syringae type III effectors, AvrRpt2 and AvrRpm1, inhibit PAMP-induced signaling and thus compromise the host's basal defense system. RIN4 is an Arabidopsis protein targeted by AvrRpt2 and AvrRpm1. The R-proteins, RPS2 and RPM1, sense type III effector-induced perturbations of RIN4. Thus, R-proteins guard the plant against type III effectors that inhibit PAMP signaling and provide a mechanistic link between the two plant defense systems. Second, nonhost resistance of Arabidopsis to P. syringae pv. phaseolicola NPS3121 (Pph) is based on multiple, individually effective layers of basal defense (Chapter 3). Arabidopsis is a nonhost for Pph, a bacterial pathogen of bean. I demonstrate that Pph elicits minimally three basal defense-signaling pathways in Arabidopsis. These pathways have unique readouts, including PR-1 expression and morphologically distinct types of callose deposition. Third, RPS2 can be activated by the corresponding effector of RPM1 (Chapter4). Expression of AvrRpm1 in the absence of RPM1 caused chlorosis and necrosis of leaves, and expression of PR-1. I constructed transgenic plants inducibly expressing AvrRpm1 in Arabidopsis containing mutations in rpm1 and additional loci involved in plant defense related signaling. Our results show that additional mutations in defense genes caused a significant reduction in AvrRpm1-induced chlorosis and PR-1 expression. I also demonstrate that AvrRpm1 weakly activate RPS2 in the absence of RPM1. AvrRpm1 induced neither symptoms nor PR-1 expression in the rpm1/rps2 plants. Thus, AvrRpm1 weakly activates RPS2. My results indicate that classical "gene-for-gene" interactions may be blurred by cross-specificity between elicitor/R protein pairs. Advisors/Committee Members: Mackey, David.

Advisors/Committee Members: Stuckey, Ronald L.

▼ Little is known about how preprophase bands (PPBs) of microtubules mark division sites, or how phragmoplasts later grow towards these sites. To more clearly define these aspects of cell division, PPBs and phragmoplasts were characterized in three dimensions in living Arabidopsis leaf epidermal cells that expressed a constitutively active alpha-tubulin-GFP construct. Contrary to classical models of cell division, both PPBs and phragmoplasts were found to form asymmetrically in that the arrays appear first in the cell cortex adjacent to the mesophyll. Cell division was also examined in tonneau2 (ton2). Despite the absence of PPBs in ton2, preprophase nuclei are properly positioned in the inner cell cortex. However, cytokinesis is incomplete and phragmoplasts are visibly disrupted, showing that TON2 is also needed for cytokinesis, not just for PPB formation. In wild-type plants, PPBs and phragmoplasts are oriented away from three different types of stomatal precursor cells, supporting the idea that intercellular signaling patterns stomata. The interphase microtubule arrays of guard mother cells and guard cells were characterized by stage. Abnormal stomatal clusters form in the too many mouths (tmm) mutant due to misplaced asymmetric divisions. PPBs and phragmoplasts in tmm are also misplaced, but otherwise normal in development and structure. This suggests that TMM helps laterally orient asymmetric divisions, but that it is not required for the asymmetric inner-to-outer development of the PPB or phragmoplast. Finally, the predictable polarity of cytokinesis was used to help define the stages of cortical microtubule re-population at the cytokinesis-interphase transition in wild-type plants. Although previous reports using cultured cells suggested that the first interphase microtubules form at the nuclear surface, I found that microtubules initiate directly in the cell cortex. These results suggest that the interphase array can arise from direct seeding of microtubules in the cell cortex. Overall this work provides an analysis of the spatial and temporal features of microtubule array development in the Arabidopsis leaf epidermis, with special attention to critical cell types and cell division. This analysis provides a framework for analyzing the cell biological features of genes that affect division, the cytoskeleton, cell wall formation, differentiation, and morphogenesis. Advisors/Committee Members: Ding, Biao.

Advisors/Committee Members: Verduin, Jacob.

Advisors/Committee Members: Stuckey, Ronald L.

Advisors/Committee Members: Transeau, E. N.

▼ Chlorophyll synthesis in angiosperms requires light because, in contrast to other photosynthetic organisms, they rely exclusively on a light-dependent mechanism to reduce protochlorophyllide during chlorophyll biosynthesis. NADPH:protochlorophyllide oxidoreductase (POR) catalyses a light-dependent reduction of protochlorophyllide to chlorophyllide, which is subsequently transformed to chlorophyll. In etioplasts during skotomorphogenesis, POR forms a photolabile aggregate of NADPH-POR-Pchlide localized to the prolamellar bodies. In Arabidopsis there is a three-member gene family encoding structurally related but differentially regulated POR enzymes denoted PORA, PORB and PORC. PORA and PORB accumulate during skotomorphogenesis. PORB and PORC accumulate during seedling development and throughout the life of the plant, during which they are responsible for bulk chlorophyll synthesis. Here I describe the detailed molecular-genetic dissection of the functions of the different POR isoforms. While single porB-1 or porC-1 null mutants display no distinct light-grown phenotypes, the porB-1 porC-1 double mutant displays a severe xantha (highly chlorophyll-deficient) phenotype. In response to illumination, chlorophyll production, thylakoid stacking and photomorphogenesis are restored in the PORA-overexpressing porB-1 porC-1 transgenic lines. Therefore, the porB-1 porC-1 double mutant is functionally rescued by ectopically expressed PORA, which suffices in the absence of either PORB or PORC to direct bulk chlorophyll synthesis and normal plant development. Using reverse genetic approaches, our lab identified a porA-1 null mutant which I have characterized here; additionally I have characterized PORA RNAi knockdown lines. The porA-1 and PORA RNAi lines display photoautotrophic growth blocks which are partially rescued on sucrose-supplemented media. porA-1 mutant seedlings display defects in etioplast development with reductions in prolamellar body accumulation and photoactive Pchlide conversion. Further analysis of the porB-1 porC-1 double null mutant reveals independent PORA catalytic activity in the cauline leaves of low-light grown plants. The porA-1 porB-1 and porA-1 porC-1 double mutants were used investigate the contributions of each POR individual isoform to Chl biosynthesis and growth in green plants. The porA-1 porB-1 and porA-1 porC-1 mutants have photoautotrophic growth defects which resemble that of the porA-1 single mutant. In addition, etioplast development in the porA-1 porB-1 double mutant is defective, with no detected prolamellar body formation or photoactive protochlorophyllide conversion. Advisors/Committee Members: Armstrong, Greg.

▼ The nuclear envelope (NE) is the distinctive attribute of eukaryotic cells. It separates chromatin from the cytoplasm, provides an anchoring surface for chromatin and is involved in organizing nuclear architecture. One of the key functions of the NE during interphase is regulation of nucleocytoplasmic transport. It also plays a pivotal role in the complex dissociation and re-association of the nucleus during open mitosis. While the NE has extensively been studied in animals, the function as well as protein composition of the NE in plants is barely understood. LeMAF1 is a plant-specific, NE-associated protein first identified in tomato. It is the founding member of a group of WPP-domain containing NE-associated proteins that includes RanGAP and the Arabidopsis WPP family, comprised of WPP1, WPP2 and WPP3. Here, the localization and function of WPP-domain proteins were investigated. WPP1 and WPP2 are targeted to the NE in a developmentally regulated fashion. Also, LeMAF1 is associated with the outer NE and the nuclear pores in interphase cells, and WPP1 is associated with the immature cell plate during cytokinesis. Besides its NE-association, LeMAF1 localizes in cytoplasmic speckles that are components of the Golgi apparatus. Further, the binding of WPP-domain proteins to the Golgi-associated, coiled-coil protein, LeWAP, was investigated. The coiled-coil domain of LeWAP is the primary interaction domain with WPP-domain proteins. RNAi lines with undetectable expression of all three Arabidopsis WPP genes at the RNA as well as protein level have shorter primary roots and a reduced number of lateral roots. This is in turn caused by reduced numbers of dividing cells. Together, these data demonstrate the first example of regulated NE targeting in plants and identify a class of plant-specific NE and Golgi-associated proteins involved in mitotic activity. The molecular functions of WPP-domain proteins and whether those functions are interconnected at the NE and Golgi yet remains to be determined. Lastly, the cloning and characterization of a transmembrane domain-containing, coiled-coil protein, TMD1, is described. The asymmetric localization of TMD1 at the anticlinal surface of the plasma membrane is actin dependent and mediated by Golgi vesicles. Arabidopsis TMD1 knockout mutant plants are highly branched and possess shorter siliques, hypocotyls and inflorescences. The latter two are caused by reduced cell elongation. Taken together, a role for TMD1 in auxin transport is postulated. Advisors/Committee Members: Meier, Iris.

Advisors/Committee Members: Stuckey, Donald.

Advisors/Committee Members: Anderson, Stanley R.

Advisors/Committee Members: Sampson, H. C.

Advisors/Committee Members: Transeau, Edgar Nelson.

Advisors/Committee Members: Transeau, Edgar Nelson.

▼ Cassava (Manihot esculenta, Crantz) roots are the primary source of calories for more than 500 million people. Cassava leaves and roots, however, contain potentially toxic levels of the cyanogenic glycoside, linamarin. Consumption of residual cyanogens in incompletely processed cassava roots can result in cyanide poisoning due to conversion of the cyanogens to cyanide in the body. Our objective was to eliminate cyanogens from cassava so as to eliminate the need for extensive food processing. To achieve this goal we generated transgenic cassava plants in which the expression of the CYP79D1 and CYP79D2 genes, that catalyze the first-dedicated step in linamarin synthesis, was inhibited. Using a leaf-specific Cab1 promoter to drive the antisense expression of the CYP79D1/CYP79D2 genes we observed up to a 94% reduction in leaf linamarin content associated with an apparent complete inhibition of CYP79D1/CYP79D2 expression. Significantly, the linamarin content of roots also was reduced by 99% in transgenic plants. These results suggest that linamarin is transported from leaves to roots. To investigate linamarin transport further we generated transgenic cassava plants in which the expression of CYP79D1/CYP79D2 was inhibited using a tuber-specific patatin promoter to drive the antisense expression of the genes. Transformants with complete inhibition of CYP79D1/CYP79D2 expression in the roots had no reduction in the root linamarin content compared to wild-type plants. Thus it is apparent that young cassava plants (3-4 month old) transport cyanogenic glucosides from the leaves to the roots. It has been demonstrated that the major cyanogen present in poorly processed cassava roots is acetone cyanohydrin and that cassava roots have substantially lower levels of HNL than leaves. We postulated that elevated expression of HNL in roots would accelerate the conversion of acetone cyanohydrin to cyanide. To test this hypothesis we have over-expressed HNL in transgenic cassava plants under the control of 2X35S CaMV promoter. The 2-fold elevation of HNL levels was correlated with substantially reduced levels of acetone cyanohydrin in homogenized root tissues. Importantly the over-expression of HNL in roots of transgenic cassava plants does not affect the steady-state linamarin levels in intact roots, thus retaining the herbivore deterrent attributes of cyanogens. Advisors/Committee Members: Sayre, Richard T.

Advisors/Committee Members: Racine, Charles.