2
Risk assessment of mercury ion heavy metal exposure on plant DNA damage using RAPD and physiological alterations in Mentha arvensis seedlings
P. Venkatachalam and R. Manikandan Department of Biotechnology, Periyar University, Salem-636011, TN, India
     Phytoremediation is a technology for extracting or inactivating various pollutants including heavy metals. Mentha arvensis is a fast growing plant with high biomass production that is common in tropical areas. However, its tolerance to heavy metals and its ability to accumulate them has yet to be investigated. Here we test the hypothesis that Mentha arvensis is able to hyperaccumulate high concentration of mercury (Hg). Plants grown hydroponically with different concentrations of Hg and has the ability to tolerate at 20 mg l-1Hg Cl2 (up to 12 days). Metal toxicity symptoms were observed at the concentration of ?20 mg l-1Hg. The exposure of Hg for a short period induced a dose dependent physiological changes as evidence by the biochemical changes, antioxidative enzyme changes, appearance and disappearance of DNA bands in the RAPD profile suggesting that the possible mechanism of Hg metal phytotoxicity in Mentha arvensis to be via oxidative stress. The root and shoot length of Mentha arvensis were gradually reduced with increasing mercury level in growth medium. A coordinated increase in enzymatic antioxidants (APX, POX and CAT) was noted with an increase in mercury concentration in the tissues. This indicates that Mentha may have a detoxification metabolism to cope with such a high concentration of mercury. As a defensive mechanism, antioxidative enzymes, especially APX, CAT and POX play an important role in scavenging AOS, suggesting strong internal detoxification mechanisms inside plant cells. The Scanning Electron Microscopy (SEM) analysis showed that the localization of mercury was mainly observed in the root xylem and in leaf tissues. The RAPD analysis is highly sensitive method for the detection of DNA damage induced by environmental pollution like mercury. The normal of missing bands enhanced with the increasing mercury concentration and the genomic template stability reflecting changes in RAPD profiles were significantly affected. The change occurring in RAPD profiles of the leaf tissues following Hg treatment presents alterations in band intensity, gain or loss of bands compared with control. The present results showed that the DNA polymorphism detected by RAPD analysis can be applied as a suitable biomarker assay for the detection of genotoxic effects of heavy metal contamination on plants. Mercury exposure significantly increased antioxidants enzyme activities and found changes at DNA level with increasing concentrations of Hg in the medium, suggesting its usefulness in phytoremediation application.

3
A Novel Cyclin Dependent Kinase in the Green Alga Chlamydomonas that Mediates Cell Size Checkpoint Control
Yubing Li, Brad Olson, Garrett Anderson and James G. Umen Plant Biology Laboratory, The Salk Institute, La Jolla, CA 92037, U.S.A. Email: yuli@salk.edu
     Proliferating cells coordinate growth and division through size checkpoints whose underlying mechanisms are not well understood. The unicellular green alga Chlamydomonas reihardtii uses size checkpoints to regulate its multiple fission cell cycle. Since cell growth and division are partially uncoupled, Chlamydomonas is uniquely suited for dissecting cell size control. The Chlamydomonas retinoblastoma (RB) tumor suppressor homolog MAT3, is a cell cycle repressor and key mediator of size control, but it is not known how MAT3/RB is regulated in response to cell size. Using forward genetic screens we isolated two mutant alleles of a gene designated CDKG1 whose deletion phenotype is large cells and which functions upstream of MAT3/RB in a linear genetic pathway. Importantly, CDKG1 mutants only affect cell size control without interruption of cell cycle kinetics. CDKG1 encodes a novel cyclin dependent kinase with a unique 90 amino acid N-terminal extension. Complementation of a yeast cdk1 temperature sensitive mutation indicated that CDKG1 retains the core functions of a cell cycle CDK. CDKG1 interacts specifically with Chlamydomonas D-type cyclins and CDKG1/D-cyclin complexes can phosphorylate MAT3/RB as a key substrate. CDKG1 mRNA and protein level were found to be cell cycle regulated. CDKG1 protein only accumulates in nucleus of mitotic cells, which indicates that CDKG1 plays an important role in S/M phase of the cell cycle. Further, transgenic strains expressing CDKG1 constitutively showed a small-cell phenotype. Together, these data suggests that CDKG1 activity is rate-limiting for size checkpoint control, and represents a new function for RB-kinases in a unicellular eukaryote.

4
Characterization of Polyphenol Oxidase in Walnut
Soha Araji, California State University San Marcos Kurt Patterson, California State University San Marcos Stephen Anderson, California State University San Marcos Matthew A. Escobar, California State University San Marcos
     The polyphenol oxidase enzyme (PPO) is almost ubiquitous in Kingdom Plantae. PPO catalyzes the oxidation of phenolic compounds to quinones, which polymerize to form brown-colored phytomelanins. Although the physiological function of PPO in plants is unclear, recent studies have suggested that PPO has potential roles in seed coat coloration, pathogen resistance, and insect resistance. Walnut (Juglans regia) generates an exceptionally diverse group of phenolic compounds in leaf and hull tissues, however, little is known about PPO in walnut. Recently, we cloned a single PPO-encoding gene from walnut and designated the gene jrPPO1. In order to investigate the physiological function(s) of this walnut PPO, we constructed transgene vectors designed to either overexpress or silence jrPPO1. Eight jrPPO1-silenced transgenic lines with PPO-activities ? 1% of wild type were identified, but none of the plants transformed with the jrPPO1 overexpression construct displayed elevated PPO activity. Using protein extracts from the leaves of wild-type and jrPPO1-silenced plants, we identified the likely phenolic substrates for JrPPO1 in planta and performed basic biochemical characterization of the enzyme. Preliminary studies suggest that PPO may play a role in defense against the causal agent of walnut blight, Xanthomonas campestris pv. juglandis, since this pathogen appears to replicate more rapidly in the leaves of jrPPO1-silenced plants than in wild-type plants. PPO also appears to have an as-yet uncharacterized role in the control of cell death, since jrPPO1-silenced lines exhibit a lesion-mimic phenotype (necrotic leaf spots) whose severity increases over the course of the growing season.

5
Auxin Biosynthesis in Agrobacterium tumefaciens
Kurt Patterson, California State University San Marcos Jacqueline Engel, California State University San Marcos Matthew Escobar, California State University San Marcos
     Indole-3- acetic acid (IAA) is involved in virtually every aspect of plant physiology. Several studies have demonstrated a role for IAA in the suppression of plant immune responses, which may explain why many plant pathogens have the ability to synthesize IAA. One of the best-studied examples of an IAA-producing phytopathogen is Agrobacterium tumefaciens, the causal agent of crown gall disease. We examined the ability of A. tumefaciens to synthesize IAA in culture, and found that A. tumefaciens releases IAA into culture supernatants (31 µg IAA/ml) when supplemented with 1 mM tryptophan. This tryptophan-dependent IAA production is independent of the Ti plasmid, since strains of A. tumefaciens lacking this plasmid retained high-level auxin production. Thus, we are currently working to identify the genetic basis of IAA production in A. tumefaciens. Several IAA biosynthesis pathways have been described in plants and bacteria, including the IAN, IAM, TAM, and IPyA pathways. Recently, the chemical inhibitor aminooxy acetic acid (AOA) was shown to inhibit PLP-dependent-aminotransferase enzymes in the TAM and IPyA pathways. The application of AOA to A. tumefaciens cultures reduced tryptophan-dependent IAA production by ~90%, suggesting a chromosomally located TAM and/or IPyA pathway in A. tumefaciens. Bioinformatic analyses of the A. tumefaciens strain C58 genome identified putative homologs of the Arabidopsis thaliana AAO1 gene and the A. thaliana PDC2 gene, both of which encode enzymes in the IPyA pathway. We are currently in the process of performing site-directed mutagenesis of these genes to define the genetic determinants of IAA biosynthesis in A. tumefaciens.

6
Genetic and Anatomical Analysis of Tassel Branch Angle in maize
Fang Bai, Robert Schmidt
     In grass species, the pulvinus is a structure found in the axil of inflorescence branches. The size and shape of the pulvinus affects the angle at which the lateral branches emerge from the main axis, and therefore has a large impact on inflorescence architecture. Through EMS mutagenesis we have identified three recessive loci in maize that affect the tassel branch angle. Mutations in these genes cause the tassel branch angle to become extremely acute. In general, mutant plants have a much smaller pulvinus than their normal sib plants. The pulvinus is composed of parenchymatic cells and enlargement of the pulvinus during development is due to cell division, not cell expansion. Two groups of mutants correspond to the previously identified genes, ramosa2 (Ra2) and liguless1 (Lg1). The third we have identified through positional cloning and encodes a TCP homolog named ZmTCP. ZmTCP is a transcription factor belonging to the plant-specific TCP family. It is expressed strongly in developing tissues, such as developing tassels and developing juvenile leaves. The translational expression of ZmTCP in developing tissues by immunolocalization overlaps with transcriptional expression by in situ hybridization. Also, the expression of ZmTCP in the developing pulvinus is consistent with the genetic analyses indicating that ZmTCP is involved in the pulvinus formation. We are progressing with our analyses to understand how Ra2, Lg1 and ZmTCP interact to promote pulvinus formation and inflorescence branch angle variation. Inflorescence branch angle also varies in other grasses such as sorghum and rice, and has become fixed in various inbred lines. We will investigate the possible conservation in this program of inflorescence development using the genes we have identified in maize.

7
Three Approaches to Enhanced Phytoremediation of Chlorpyrifos
Keum Young Lee (1), Stuart E. Strand (1, 2), and Sharon L. Doty (1) (1) School of Forest Resources, College of the Environment, University of Washington; (2) Department of Civil and Environmental Engineering, University of Washington, Seattle, WA 98195, USA. *E-mail: ky1206@uw.edu
     Chlorpyrifos (CPS) is an organophosphorus insecticide that is implicated in environmental and human health problems. These problems may be solved by the emerging phytoremediation technology, which is the use of plants for the cleanup of environmental contaminants. Here we introduce three approaches to enhanced phytoremediation of CPS. To evaluate plant potential for uptake of CPS, several plant species of poplar and willow were investigated. Analysis of the CPS removal showed that CPS can be taken up by plants. Significant amounts of CPS accumulated in plant tissues, and CPS did not persist in the tissues, suggesting further metabolism. To our knowledge, this work represents the first report for phytoremediation of CPS using poplar and willow. The second approach is phytoremediation of CPS by transgenic poplar. Transgenic expression of genes involved in CPS metabolism is expected to increase removal of CPS. Cytochrome P450s (CYP) activate CPS, forming CPS-oxon, which paraoxonase 1 (PON1) detoxifies. Human CYP2B6 and rabbit PON1 have been cloned into plant expression vectors and poplar has been transformed. Selected transgenic lines will be investigated for CPS uptake and degradation. The third approach is the use of plant-derived PON1 protein for rapid on-site remediation of spills as a soil amendment. In order to express the PON1 protein at high levels in plants, rabbit PON1 has been cloned into the chloroplast transformation vector and a tobacco clone has been transformed using a particle delivery system. The transgenic tobacco will be analyzed for PON1 protein activity assay and maximum production of PON1.

8
The dueling roles of the plant leucine aminopeptidases: exopeptidases or molecular chaperones?
Melissa A Scranton, Ashley Yee, and Linda L Walling Department of Botany and Plant Sciences and Center for Plant Cell Biology University of California, Riverside
     In plants, there are two classes of leucine aminopeptidases: LAP-N and LAP-A. Both LAP-N and LAP-A are hexameric exopeptidases. While LAP-N is expressed constitutively in all plants, LAP-A has only been identified in solanaceous species and is induced by a wide variety of biotic and abiotic stresses in tomato (Solanum lycopersicum). SlLAP-A is important in insect deterrence in tomato and has regulatory role in the late branch of wound signaling. However, there is currently no known mechanism for SlLAP-A's function. Studies from other organisms have shown that some aminopeptidases can be bifunctional, acting as peptidases and molecular chaperones. Three biochemical assays were used to determine if plant LAPs could act as molecular chaperones and protect model protein substrates from heat-stress induced damage. These assays measured the ability of LAP to prevent protein unfolding, prevent protein aggregation and promote protein refolding. These assays indicated that the tomato SlLAP-A and SlLAP-N and the Arabidopsis AtLAP1 and AtLAP2 are molecular chaperone in vitro. The molecular chaperone activities of mutant SlLAP-A proteins that were aminopeptidase deficient or could not assemble into a hexameric structure (and therefore also aminopeptidase deficient) were also tested. SlLAP-A's chaperone activity was independent of its peptidase activity and disruption of SlLAP-A's hexameric structure increased chaperone activity. These data shed new light on the complexity of plant LAP proteins and suggest new potential roles for SlLAP-A in defending tomato against stress.

9
Monitoring reconfiguration of cellular metabolism in response to hypoxia in Arabidopsis thaliana by NMR-based metabolomics
Kayla A. Kaiser (1,2), Cristina Branco-Price (2), Angelika Mustroph (2), Cynthia K. Larive (1), and Julia Bailey-Serres (2) (1) Department of Chemistry (2) Department of Botany and Plant Sciences, University of California, Riverside, CA, 92521, USA
     Metabolomics records a snapshot of the molecular phenotype of a cell, organ, or organism. Metabolic profile comparisons reveal the induction of bottlenecks and shunts in highly-branched pathways following environmental stimuli. The response to oxygen deprivation can be monitored in a model plant, such as Arabidopsis, by unified analysis of quantitative transcriptomic, translatomic and metabolomic data to form a multi-level perspective of acclimation strategies. Arabidopsis thaliana (Col-0) seed were germinated on solid media fortified with MS salts and 1% sucrose. Stress was applied to 7-d-old seedlings by replacing air with argon for 2 h or 9 h. Additionally, a 9 h stress group was allowed 1 h reoxygenation. Whole seedlings were harvested, homogenized and lyophilized prior to extraction and characterization by 1H-NMR. Roots and shoots of 2 h control and stressed groups were collected to evaluate organ-specific responses. Lactate increased in all stress samples, with a more dramatic accumulation in shoots. Alanine, thought to serve as a carbon and nitrogen sink, increased to a greater extent in shoots and remained elevated after reoxygention. Depletion of aspartate and glutamate was more dramatic in the root than the shoot, and remained low after reoxygention. Elevation of GABA occurred predominantly in roots, emphasizing distinct rearrangement of cellular metabolism in roots and shoots. The dynamic metabolic reconfiguration was generally consistent with changes in translated mRNAs. Solid phase extraction coupled to LC-MS monitoring of secondary metabolites is underway.

10
Evolution of SPARSE INFLORESENCE1 in Grasses
RoseMary Puhr and Simon Malcomber, Department of Biological Sciences, California State University – Long Beach, Long Beach, CA 90840 USA. Email: rpuhr@csulb.edu
     The YUCCA gene family catalyzes a highly conserved pathway for production of the growth hormone indole acetic acid (IAA), the predominant form of auxin in plants. YUCCA genes function in the tryptophan-dependent pathway where they catalyze the conversion of tryptamine (TAM) intermediate to N-hydroxyl TAM. YUCCA genes are functionally redundant in Arabidopsis but have non-redundant roles in grasses and petunia. SPARSE INFLORESCENCE1 (SPI1) is critical for maize reproductive development (Gallavotti et. al. 2008. Proc Natl Acad Sci USA 105, 15196-15201) but may be less important in rice (Yamamoto et al. 2007. Plant Physiol 143, 1362-1371). These data suggest SPI1 function and the extent of YUCCA gene redundancy may have changed during the diversification of the grass family. My project investigates the molecular and functional evolution of SPI1 in grasses and immediate relatives. SPI1 orthologs are restricted to monocots. SPI1-like genes are syntenic within grasses and qRT-PCR expression analyses indicate they are expressed broadly throughout various organs within the plant, though to varying degrees. In situ hybridization analyses in grasses suggest SPI1 genes have localized expression patterns that coincide with sites of local auxin biosynthesis associated with lateral branching.

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The evolution of DEF-GLO heterodimerization in the grasses
Madelaine Bartlett and Clinton Whipple
     The ABC model of floral development proposes that there are three classes of transcription factors that control floral organ identity: the A, B, and C class MADS box genes. The A class genes specify sepals, A and B class genes together confer petal identity, B and C class genes specify stamen identity and C class genes control carpel development. There are two main lineages of B class genes: the DEFICIENS/APETALA3 (DEF/AP3) lineage and the GLOBOSA/PISTILLATA (GLO/PI) lineage. In both monocots (the grasses Oryza and Zea) and eudicots (Antirrhinum, Arabidopsis, Petunia, Papaver, and Aquilegia) GLO and DEF orthologs bind DNA as obligate heterodimers. In Lilium (Liliales) and Phalaenopsis (Orchidales), however, B class homodimers were found to be capable of binding DNA. In Lilium, GLO homologs form functional homodimers while in Phalaenopsis both DEF-like and GLO-like homodimers are capable of binding DNA. We present data showing that in a close grass relative, Joinvillea, a GLO ortholog can function both as a homodimer and as part of a DEF-GLO heterodimer. Taken together, these results imply that the DEF-GLO obligate heterodimer relationship evolved separately in eudicots and monocots. We propose an investigation into the evolution of heterodimerization in the B class gene lineage, particularly in the grasses. We plan to investigate dimerization capabilities of B class genes in a number of grass and outgroup taxa. Through domain-swap experiments, selection tests, and site-directed mutagenesis we hope to identify precisely which amino acid changes were involved in the evolution of the obligate DEF-GLO interaction in grasses.

12
Plant-derived Virus Like Particles.
Dr. Tsafrir Mor, Arizona State University Sarah Kessans, Arizona State University Mark Linhart, Arizona State University
     The goal of this research is an easily scalable, affordable vaccine against HIV. The HIV protein Gag, a structural protein, was stably expressed in Nicotiana benthamiana utilizing an Agrobacterium based system. These transgenic plants were then subsequently transiently infiltrated with a deconstructed TMV expression system (ICON) to also express the HIV envelope protein gp41. Through biochemical assays, the resultant plants showed co-localization of gp41 and Gag as well as the formation of virus like particles (VLPs). The immunogenicity of plant-derived VLPs will be tested in experimental animal models.

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High-throughput Screening Genome-wide Targets of CCA1 and LHY Using an Ethanol Inducible System
Sheen X. Lu, University of California, Los Angeles Stephen M. Knowles, University of California, Los Angeles Elaine M. Tobin, University of California, Los Angeles
     The circadian clock is an endogenous mechanism that coordinates biological processes with daily and seasonal changes in the environment and confers enhanced fitness on organisms ranging from prokaryotes to humans. Circadian rhythms are generated by endogenous central oscillators that respond to input from the environment and regulate rhythmic outputs. Using an ethanol inducible system, we demonstrated that CCA1 and LHY reset the phase of the circadian clock in Arabidopsis after a transient increase in their expression, indicating that CCA1 and LHY are central oscillator components. To understand the details of how CCA1 and LHY function in generating circadian rhythms as well as identifying novel clock components, we have used microarray analysis to identify genes whose expression is affected after a transient induction of CCA1 or LHY expression. The identified targets include known clock-regulated genes and previously unknown genes, encoding transcription factors, chromatin proteins, and proteins involved in metabolism and signal transduction. Among the target genes, one of them encodes a Jumonji C (JmjC) domain-containing protein, designated JMJ30. JmjC domain-containing proteins have been shown to be involved in chromatin remodeling, acting as histone demethylases. Our results indicate that JMJ30 regulates the pace of the circadian clock in a close association with the central oscillator and reveal the importance of histone methylation in the circadian clock mechanism. In addition, the use of the ethanol inducible system provides a means for experimentally testing the effect of a pulse of virtually any protein.

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Analysis of Perturbations in Expression Correlation (APEC) Untangles Genetic Redundancy in the Arabidopsis Circadian Clock
Colleen J Doherty, University of California, San Diego Division of Biological Sciences, Center for Chronobiology Steve A Kay, University of California, San Diego, Division of Biological Sciences,
      Identifying the function of a gene of interest is often complicated in multi-cellular eukaryotes by gene duplications. In Arabidopsis 65% of genes belong to multi-gene families, the majority of these families have five or more members. This genomic redundancy is reflected in the components of the Arabidopsis circadian clock, generating a complicated network that presents a challenge to investigate. However, understanding the function and output regulation of the circadian clock is critical to improving plant growth and fitness. To overcome the analysis challenges generated by the high-levels of redundancy we take advantage of existing expression data generated by the Arabidopsis community and apply a unique twist on co-expression analysis to reveal experiments where co-expression is perturbed.
By identifying experiments where expression correlation between two family members is lost this analysis has the potential to identify experiments that may provide insights into both regulation and function of genes that otherwise appear redundant.
Using this technique we identify a potential novel regulator of circadian output. Biological validation of these results are ongoing. While this experiment focuses on identifying components of the Arabidopsis circadian clock, we anticipate that this technique will be broadly applicable for identifying both function and regulation in many systems.

15
CLE genes may be involved in multiple signaling cascades that regulate development and activities in an array of plant tissues and cells
Ling Meng and Lewis J. Feldman Department of Plant and Microbial Biology, 111 Koshland Hall, University of California Berkeley, CA 94720-3102
     Intercellular communication and coordination between adjacent cell populations are critical in cell-fate specification and cell activity, especially in meristem organization and maintenance in plants. CLE, which is the term for the CLV3/ESR-related gene family, is thought to participate in CLAVATA-WUSCHEL (CLV-WUS) and CLV-WUS-like negative feedback signaling loops to regulate shoot apical meristem (SAM) and/or root apical meristem (RAM) activity in plants. Here we show that over-expression of CLE14 or CLE20 triggers early termination of the RAM in a CLAVATA1 (CLV1)-independent, but CLAVATA2 (CLV2)-dependent manner. Additionally, we show that an exogenous application of CLE14 or CLE20 peptide inhibits, irreversibly, root growth by reducing cell division rates in the RAM in Arabidopsis. Although some CLE genes are found to express specifically in the SAM and/or RAM, many CLE genes appear to express in the tissues outside meristems in Arabidopsis. In this regard, we show that CLE14 and CLE20 express in specific tissues/cells, including highly differentiated cells, and at different developmental stages, and that over-expressing CLE14 or CLE20 also affects the development of an array of tissues/cells outside the RAM or SAM, which are consistent with its expression pattern in Arabidopsis. These results suggest that endogenous CLE genes may be involved in regulating the development and activities of multiple tissues/cells, which include (but is not limited to) the cells in the SAM and RAM of plants. These results also imply that CLE genes may participate in other signaling cascades in addition to the CLV-WUS and CLV-WUS-like pathways in plants.

16
Phylogenomics and molecular evolution of NODULIN26-like intrinsic proteins in plants
Jonathan Mares and Simon Malcomber, CSULB
     The Nodulin26-like intrinsic protein (NIP) aquaporins represent a large and functionally diverse plant specific gene family. NIPs have been shown to regulate transport of diverse substances with Nodulin26, the most comprehensively studied NIP to date, shown to facilitate transport of glycerol, formamide and ammonia. Tasselless 1 (TLS1), identified in maize mutants by Paula McSteen (University of Missouri) exhibits severely truncated tassels, is a member of the NIP group of aquaporins. Bayesian phylogenetic and comparative genomic analyses indicate that TLS1 forms a distinct, well supported monophyletic group comprised of both grasses and core eudicots, suggesting an origin for the lineage at least near the base of flowering plants and potentially deeper within the vascular plant clade. Two main domains govern Aquaporin transport activity in NIP aquaporins: 1) the dual NPA motif, and 2) the aromatic/R pore. The TLS1 clade shows a difference from other NIP aquaporins with the dual NPA motif exhibiting a completely conserved NPS, NPV motif. The aromatic/R pore and alanine (A), isoleucine (I), glycine (G) and arginine (R) residues of the pore region are all highly conserved within the TLS1 clade suggesting gene function is likely conserved within the bulk of angiosperm species. The discovery that TLS1 co-ortholog in Arabidopsis thaliana (AtNIP5;1) functions in boric acid transport implicates boron uptake as one of the broad functions of TLS1 and suggests that the mutant tassel phenotype of maize tls1 mutants is likely caused by a boric acid transport deficiency.

17
Evolution of RAMOSA3-like genes in grasses (Poaceae)
Simon Malcomber, and Chelsea Hope, Christopher Lopez Department of Biological Sciences, California State University, Long Beach, 1250 Bellflower Boulevard, Long Beach, CA 90840 USA
     The three RAMOSA genes in maize, RAMOSA1 (RA1), RA2 and RA3 are essential for the formation of short branch (or spikelet pair) meristems, with RA2 and RA3 acting as upstream regulators of RA1. Maize has two RA3-like genes – ZmRA3 and SISTER OF RA3 (ZmSRA) on chromosome 7, whereas the syntenic regions in rice and brachypodium have only a single gene that is most similar to ZmSRA.
Phylogenetic analyses show that the gene duplication events that produced the RA3/SRA and RA3-LIKE1 (RA3L1), RA3L2, RA3L3, RA3L4 and RA3L5 lineages all occurred prior to major diversification of grasses with RA3 orthologs estimated to have been subsequently lost near the base of the rice, wheat and bamboo lineage (BEP clade). ZmRA3 expression is restricted to a cup-shaped region subtending the spikelet pair meristems and a stripe between the upper and lower florets, whereas ZmSRA is expressed broadly throughout the plant with the highest expression in roots. Expression analyses of SRA and RA3 in other grasses reveal a complex pattern of evolution. SRA orthologs are expressed at the base of long branches in rice, barley, and sorghum and in vascular tissue in teosinte. RA3 expression was detected subtending long and short branch meristems and between the upper and lower florets in sorghum and teosinte and on the adaxial surface of florets in chasmanthium. Together these data suggest the restricted pattern of RA3 expression detected in maize subtending short branch meristems did not coincide with the evolution of these structures within panicoid grasses, but appears to have evolved recently, potentially during the domestication of maize.

18
Improvements on the Production Platform for Plant-Produced Butyrylcholinesterase
Neil E. Robbins II, Latha Kannan, and Tsafrir S. Mor School of Life Sciences and The Biodesign Institute, Arizona State University, Tempe, AZ 85287
     Organophosphorus compounds (OPs), such as the chemical warfare nerve agents have harmful effects
in mammals through their ability to irreversibly inhibit cholinesterase enzymes at the neuromuscular
junction. These enzymes act to terminate nerve signals by hydrolyzing the neurotransmitter
acetylcholine, and inhibition of this process leads to a “cholinergic crisis,” a potentially fatal condition.
Because of their high affinity to OPs, cholinesterases can act as scavengers and their administration
may allow the sequestration of these toxicants and curbing of exposure symptoms. To this end, a
production platform based on Nicotiana benthamiana plants has been devised. A plant-expression
optimized gene encoding human butyrylcholinesterase (BChE) has been synthesized and expressed
transiently using a deconstructed tobacco mosaic virus vector (the MagnICON system). In order to
improve accumulation of the recombinant enzyme, we have targeted the protein for several subcellular
domains including the cytoplasm, the ER membrane and the ER lumen. Our findings indicate that
highest level of accumulation is observed when protein is targeted to the ER lumen by a signal peptide.
While the endogenous human signal peptide is recognized, it is nonetheless outperformed by a bona-
fide plant signal peptide. Our efforts led to a 200-fold over the original cDNA clone of the BChE
gene in N. benthamiana, allowing for more efficient production of the enzyme for use in downstream
purification toward therapeutic use.

19
The 3’-untranslated region of the tobacco Extensin gene increases transgene expression by reducing readthrough transcripts level in N. benthamiana.
Sun Hee Park, Hugh S. Mason.
     For efficient expression of foreign genes in plants, a good expression vector system using appropriate cis-acting elements is required. In this study, we evaluated the tobacco Extensin (Ext) gene 3’ untranslated region (UTR) to mediate transcription termination and polyadenylation of foreign gene in N. benthamiana. Using transient expression, we found that the Ext 3’ UTR is the most effective in supporting green fluorescent protein and Norwalk virus capsid protein expression compared to previously established terminators. The enhanced protein expression was correlated with increased mRNA accumulation. Moreover, the level of readthrough transcripts, which could lead post-transcriptional gene silencing, was dramatically decreased by using Ext 3’ UTR. These data indicate that the Ext 3’ UTR is efficient at mRNA 3’ end formation, contributing to increased mRNA and expression levels in plants.

20
HISTONE METHYLATIONS ASSOCIATED WITH CHANGES IN GENE EXPRESSION DURING SENESCENCE
Judy Brusslan, Ana Rus, Judd Rice1. and Michael Hitchler2. Department of Biological Sciences, California State University, Long Beach, 1250 Bellflower Blvd., Long Beach, CA 90840-3702 1.Biochemistry and Molecular Biology, USC, Norris Cancer Center, 1450 Biggy St., Los Angeles, CA 90033 2. Radiation Oncology Department, Kaiser Permanente, 4950 Sunset Blvd., Los Angeles, CA 90027
     Major changes in gene expression accompany leaf senescence. Towards understanding how epigenetic mechanisms contribute to these gene expression changes, two histone modifications were measured on a genome-wide basis using ChIP-Seq. Nuclei were isolated from green leaves from 1) non-senescent plants that were 23 days old and 2) senescent plants that were 52 days old. Antibodies that recognize H3K4me3, a histone methylation associated with gene activation, or H3K27me3, a histone methylation generally associated with gene silencing, were used for chromatin immunoprecipitation. An antibody that recognizes all forms of histone3 was used as a normalization control. Immunoprecipitated DNA fragments were sequenced using the high-throughput Illumina platform, and displayed using Integrated Genome Viewer. Gene expression changes for selected genes were measured by real-time qPCR using RNA isolated from the same tissue samples. Some genes displayed expected patterns of histone modifications. For instance At1g13340, which was up-regulated 9.7x in our 52d sample, displayed an increase in H3K4me3 in the older tissue, while At2g10940, which is strongly down-regulated in older tissue, showed a decrease in H3K4me3 in the older tissue. Numerous other genes showed unexpected results. The most striking were At1g73220 and At5g45890 (SAG12) which respectively displayed 10,000-fold and 30,000-fold increases in mRNA levels in the older tissue. Despite these dramatic increases in gene expression, H3K4me3 levels remained low at both 23 and 52 days. After analyzing a larger number of genes that were up- or down-regulated during senescence, the only emergent pattern was that repressed genes displayed a loss of H3K4me3 in older tissue.

21
Senescence-Associated Intra-Organelle Protein Degradation within Isolated Chloroplasts from Arabidopsis thaliana
Scott Vande Wetering, Robert Hoiness, Travis Lee, Keykhosrow Keymanesh, and Judy Brusslan Department of Biological Sciences, California State University Long Beach
     Chloroplasts contain over 50% of cellular nitrogen, and thus degradation of chloroplast proteins is a major source of nitrogen that is remobilized during senescence. Previous work demonstrated that degradation of Rubisco large subunit (LSU) and chloroplast glutamine synthase (GS2) does occur in isolated pea chloroplasts, suggesting that intra-organelle protein degradation contributes to nitrogen remobilization. The aim of this study was to determine if intra-organelle protein degradation occurs in chloroplasts isolated from mature and senescent Arabidopsis leaves. Chloroplasts were purified on Percoll gradients, incubated for varying amounts of time, then re-purified to ensure protein degradation took place within intact organelles. In mature leaves LSU was found to be completely stable even when incubated for three days, however GS2 was almost completely degraded within four hours. In senescent chloroplasts, isolated from tissue that contained 50% chlorophyll, similar degradation patterns were noted. In addition, Rubisco Acitvase (RCA) was almost completely degraded after 24 hours. Chloroplasts were also isolated from the s33Ab mutant, which encodes a serine protease/lipase that is up-regulated 10-fold in senescent tissue. Chloroplasts isolated from senescent s33Ab leaf tissue displayed rates of LSU, GS2 and RCA degradation similar to wildtype. These data suggest that intra-organelle degradation of LSU is not contributing to nitrogen remobilization, however degradation of GS2 and RCA does occur rapidly in the chloroplast, and could contribute to N remobilization.

22
Characterization of a Butyrylcholinesterase (Y332S) produced in Nicotiana benthamiana With the Possibility of being Reactivated by an Oxime
Matthew T. Hilton (1), Latha Kannan (1), John R. Cashman (2), Jun Zhang (2),and Tsafrir S. Mor (1) (1) School of Life Sciences and The Biodesign Institute, Arizona State University, Tempe, AZ 85287 (2) Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121
     Organophosphorous compounds (OPs) inhibit the human enzyme acetylcholinesterase (AChE) which leads to excessive accumulation of the neurotransmitter acetylcholine. This accumulation leads to overstimulation of the cholinergic circuitry in the central nervous system and in muscles, potentially leading to paralysis and death. The homologous human enzyme, butyrylcholinesterase (BChE), is found in serum and is believed to function in clearing low-levels of environmental toxicants. The enzyme is a useful scavenger of OPs but because of the 1:1 stoichiometry of its function is required in large doses to be effective. This enzyme can be produced in a plant platform, Nicotiana benthamiana, using a deconstructed tobacco mosaic virus to transiently express the protein. The ability to scale up quickly, be cost effective, and have no possibility of human pathogens of other platforms makes expressing BChE in plants an excellent alternative source of BChE instead of human blood. To make scavenging even more efficient, potentially turning BChE we are working on producing BChE variants that will be able to reactivate after binding OPs, either spontaneously or with the help of an oxime molecule. Unlike normal human BChE, the BChE Y332S variant identified through molecular evolution can be reactivated by oximes. The gene encoding this variant was expressed in N. benthamiana and has been partially purified. Future studies will include tests the activity against a panel of cholinesterase inhibitors, along with the use of an oxime to test for reactivation of the enzyme.

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Identification of the cis Regions Mediating High Light Responsiveness in the Arabidopsis thaliana ELIP1 Promoter
Daisy Flores, Ana M Rus Alvarez, Francisco Rodriguez, Judy A Brusslan. Department of Biological Sciences, California State University, Long Beach, 1250 Bellflower Blvd. CA 90840 USA
     The Early Light Inducible Protein (ELIP) is expressed in response to high light (HL) intensities and degraded after the plant is returned to low light. In Arabidopsis, the ELIP1 gene (At3g22840) is more sensitive to HL than ELIP2 (At4g14690), and it is proposed to down-regulate chlorophyll synthesis during HL. This study is aimed to identify cis regions of the ELIP1 promoter using site-directed mutagenesis in known light regulatory elements and a novel 12 bp element found to be unique to both ELIP1 and ELIP2. Mutations were made in the following elements: TCAATA at -118 (CAAT), GCGCGA at -136 (GT1-like element), CGGTTC at -152 (MYB core), TTACCT at -145 (TATA box 5), CACGTG at -170 (G-box), AGATAG at -202 (GATA), GAGACT at -229 (GT1 box), AGGCCACGCCAT, at -668 (12 bp element) and CTACGTGT at -549 (Upstream G-box). Promoter activity was measured indirectly by the GUS (b-glucuronidase) assay in transgenic plants (n > 20) expressing the GUS gene under the control of either the non-mutated promoter (984 bp control line) or the promoter with mutated putative cis regions. Significant differences were observed between the 984 bp control line and the 12bp single mutant, as well as the following double mutants: 12bp-UpGbox, 12bp-Gbox, and UpGbox-Gbox and the triple mutant 12 bp-Gbox-UpGbox (p < 0.001; One-ANOVA). These data suggests that the 12 bp cis region, shared by both ELIP1 and ELIP2, in combination with the two G-boxes, Gbox and UpGbox, is important for the regulation of the ELIP1 gene under high light conditions.

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