Lemna gibba was used as a model aquatic angiosperm to study the effects of ethylene glycol. Ethylene glycol, usually thought of as a benign pollutant, was shown to cause a phenomenon that was termed the polyol effect. Poly(ethylene glycol)s with carbon chain lengths of less than 34 residues, as well as two ethers of ethylene glycol, also elicited the polyol effect Although ethylene glycol was not extremely toxic (acute toxicities in the mM rather than muM or nM range), it interacted synergistically with other organic chemicals to tentiate their toxicities. Thus, the toxicity of naphthalene was enhanced in the presence of ethylene glycol but, due to the volatility of naphthalene, experiments to demonstrate that ethylene glycol also enhanced the metabolism of naphthalene were inconclusive A spectroradiometric method was devised to quantify the polyol effect. The threshold concentration of ethylene glycol at which the polyol effect could be detected by this method was between 35 and 40 mM. The earliest developmental time for the detection of ethylene glycol stress was between the second and third generation of daughter fronds. The spectroradiometric methods that were developed for measuring ethylene glycol stress were shown to be considerably more sensitive and reproducible than any other available method. Spectroradiometry, using the duckweeds as bioindicators, could provide a relatively quick, sensitive monitoring technique for aquatic pollution of various kinds The toxicities of thorium and cadmium were not potentiated by ethylene glycol and neither were their uptakes. Both control and ethylene glycol-grown fronds absorbed thorium. Only a small portion of the absorbed thorium was leachable indicating that thorium uptake was either a result of active cellular uptake or binding to external cell surfaces. However, thorium absorption by dead L. gibba fronds was equally fast showing that uptake was a result of biosorption rather than active cell processes. DRIFTS analysis of thorium-loaded fronds revealed that thorium was bonded to carboxylic sites. Sugar cane bagasse was also able to remove thorium from an aqueous medium suggesting that plant materials in general could be highly effective absorbents for the remediation of actinide pollution / acase@tulane.edu

The inevitable fate of much of the ethylene glycol used for deicing bridges, aircraft and airport runways, and from leaking and overheating automobile radiators is as runoff to adjacent surface waters which commonly contain higher plants, including members of the duckweed family, the Lemnaceae. However, ethylene glycol is usually thought of as being a relatively benign pollutant and therefore its effects on higher plants have received little attention. The EC50 for ethylene glycol with respect to the inhibition of frond reproduction in axenically-grown Lemna gibba is 176 mM. HPLC and GC/MS studies indicate that ethylene glycol is not metabolized by duckweed. However, after having grown in the presence of ethylene glycol, the fronds of L. gibba are a darker green, translucent color, tend to sink, and generated gas bubbles in their growth media. It is hypothesized that these effects are due to a disruption by ethylene glycol of the pectin layer between cells as evidenced by the appearance of intercellular gaps in the aerenchymatous tissues. Other polyols, including propylene glycol and glycerol, produced the same effects as ethylene glycol. The result of the creation of the intercellular gaps is to increase the uptake of solutes and water from the growth media into the intercellular air spaces. The enhanced uptake of water caused the fronds to sink as well as change the optical properties which resulted in the darker green appearance. The enhanced uptake of nutrients led to the stimulation of growth at concentrations of ethylene glycol below 80 mM. The enhanced uptake of sucrose led to enhanced metabolism and an increased evolution of carbon dioxide as reflected by the bubbles in the growth media. However, the enhanced uptake of organic and inorganic pollutants led to their enhanced toxicities. Therefore, though ethylene glycol may be of relatively low direct toxicity, it can through various interactions, potentiate the toxicities of other pollutants / acase@tulane.edu

A 712 bp partial cDNA clone (czbp- 1) of the citrus vascular zinc binding protein (CVZBP) was isolated using reverse transcriptase polymerase chain reaction (RT-PCR). The deduced amino acid sequence of czbp-1 was identical to the N-terminal amino acid sequence for the CVZBP. Czbp- 1 had a 549 bp open reading frame and two putative polyadenylation sites, +20 bp and +103 bp relative to the poly-A tail. The deduced amino acid sequence had identity with members of the Kunitz soybean proteinase inhibitor (KSPI) family. Many members of this family are present in high concentrations in storage organs such as seeds and tubers, increase in response to abiotic stress, and are considered defense or stress response proteins. The CVZBP did not appear to fit in this category. Unlike many members of the KSPI family CVZBP was not detected in citrus seeds and protein levels decreased in response to wounding. Transcript also decreased in response to osmotic stress; a similar result previously was reported for CVZBP protein levels. Accumulation of CVZBP and its transcript increased in Zn deficient citrus seedlings compared to those receiving sufficient levels of Zn, indicating that Zn nutrition can modulate CVZBP expression. Recombinant CVZBP was produced and used to determine the capacity of this protein to inhibit several types of proteinases. The CVZBP inhibited the cysteine proteinase, papain, but not the serine proteinases, trypsin and chymotrypsin. CVZBP protein was immunolocalized primarily to the xylem parenchyma in vascular tissue of citrus midribs. Based on these results it is possible that the CVZBP has a function in vascular differentiation. Cysteine proteinases were identified in developing tracheary elements in Zinnia cell cultures. Addition of inhibitors of cysteine proteinase to these cultures prior to secondary cell wall deposition prevents differentiation of the cells into tracheary elements. Perhaps cysteine proteinase inhibitors, such as the CVZBP, in the xylem, contribute to timing of tracheary element differentiation and determination.

The goal of my research was to better understand the mechanism of plant cold acclimation using Arabidopsis thaliana as a model system. Our genetic screening method utilizes a line of transgenic Arabidopsi plants harboring the firefly luciferase reporter gene (LUC) fused to the RD29A promoter (RD29A-LUC), which contains the cold- and osmotic stress-responsive DRE/ CRT element and the ABA-responsive ABRE element (Ishitani et al., 1997). The RD29A-LUC plants were mutagenized with ethylmethane sulfonate and mutants with aberrant luminescence responses were selected from the resulting M2 population by luminescence imaging using a cooled CCD camera (Ishitani et al., 1997). hos2-1 (for high expression of osmotically responsive genes) mutant was isolated based on enhanced expression of RD29A and other stress genes under low temperature treatment (Chapter 2). Enhanced gene expression was observed only in response to cold stress, not to osmotic stress or ABA, in hos2 mutants. Compared with the wild-type plants, the hos2-1 mutant plants are less capable of developing freezing tolerance when treated with low non-freezing temperatures, indicating that HOS2 is a negative regulator of low temperature signal transduction important for plant cold acclimation. Chapter 3 characterized the function of the HOS1 gene that was found to encode a novel protein with a RING (Really Interesting New Gene) finger motif near the amino terminus. The observation that hos1 plants are more sensitive to freezing temperatures even though they have enhanced expression of CBF transcription factors and of their downstream cold responsive genes suggests that controlling the stability of critical proteins in the cell is important in the cellular response to environmental stimuli. In contrast to hos1 and hos2 mutants, the los2 mutant was identified by its reduced expression of the RD29A-LUC transgene under cold treatment but not ABA, salt or PEG treatments (Chapter 4). LOS2 is critical for chilling as well as freezing resistance. The los2 mutation specifically impairs cold regulated expression of the endogenous COR/KIN/RD/LTI genes. The LOS2 gene encodes an enzyme, enolase, which converts 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway. Several lines of the evidence for possible LOS2 function as a transcriptional regulator are presented.

Elongation factor 1A is one of the lysine-rich proteins increased in o2 mutants, and its concentration is highly predictive of the protein-bound lysine content of the endosperm. Understanding the biological basis of this relationship could help to explain the mechanisms of lysine accumulation in the endosperm, providing new insights for developing maize genotypes with better nutritional quality. Three different eEF1A isoforms were purified from developing endosperm and investigated in their accumulation, structural and functional activities. The accumulation of the isoforms appears to be developmentally regulated and independent of the o2 mutation. The purified proteins differed in their ability to bind F-actin in vitro, suggesting they are functionally distinct. The isoform that binds actin most effectively was the most predominant in high eEF1A genotypes, which may be related to enhanced cytoskeleton formation, and therefore increased synthesis of cytoskeleton-associated proteins in these genotypes. Tandem mass spectrometry revealed each isoform is composed of the four same gene products, which are modified post-translationally by methylation and phosphorylation. The chemical differences that account for their different actin binding activities could not be determined. Recombinant inbred lines varying in eEF1A content were developed from a cross between a high (Oh51Ao2) and a low (Oh545 o2) eEF1A inbred. The parental inbreds and RILs with the highest and lowest eEF1A content were used to investigate patterns of gene expression and protein synthesis. Transcript profiling with an endosperm EST microarray identified about 110 genes coordinately regulated with eEF1A. These genes encode proteins involved in several biological structures and processes, including the cytoskeleton, the endoplasmic reticulum and the protein synthesis apparatus. The content of alpha-zein and several cytoskeletal proteins was measured in high and low eEF1A inbred lines, and the levels of these proteins were found to correlate with that of eEF1A. Thus, higher levels of eEF1A may be related with a more extensive cytoskeletal network surrounding the rough ER and increased translation of mRNAs encoding cytoskeleton-associated proteins, all of which contribute significantly to the lysine content of the endosperm.

The architecture of a plant is intimately tied to its fitness. Knowledge of the processes and patterns of growth form evolution can therefore contribute to a richer understanding of plant evolution. The genus Adenia (Passifloraceae) of ca. 100 species is an Old World lineage in which growth form radiated. I constructed a molecular phylogeny of the group, analyzed the stem and tuber anatomy of over half the species, and investigated patterns of growth form evolution in a phylogenetic context. I also described four new species and a new combination. Predictions based on evolutionary developmental models of growth form evolution were tested in Adenia, and one of them, the homeotic switch hypothesis, was tested throughout the eudicots. The switch hypothesis claims that the storage tissue of tubers and stems results from a common developmental origin. Phylogenetic analyses revealed that growth form transitions were frequent, and anatomical studies revealed traits that are associated with each growth form; moreover, traits are shared between tubers and succulent stems as predicted by the switch hypothesis. As expected, tuberous plants and succulents are also closely related across the eudicots. The switch hypothesis is substantiated in Adenia and the eudicots as a whole.

Endoreduplication is a process of genome duplication without mitosis. Although endoreduplication is common among plants and animals, the molecular mechanisms involved with this process are not fully understood. Two strategies were used to determine the genetic components involved with endoreduplication. One, based on QTL analysis and the other strategy involved identifying cell cycle candidate genes, and characterizing them in regard to endoreduplication. To map genes influencing endoreduplication, four backcross populations were created from crosses between a high (Sg18) and a low (Mo17) endoreduplication inbred and their F1 (Sg18 x Mo17) progeny. In all, fourteen quantitative trait loci were identified that affect the degree of endoreduplication in maize endosperm. Six QTLs were mapped with a triploid mode of inheritance in the endosperm. Two QTLs were mapped with parent-of-origin effect inheritance. Six QTLs were mapped using a model that considers genetic interaction between embryo and endosperm. Previous studies with maize endosperm showed that accumulation of a cyclin-dependent kinase (CDK) inhibitor is coincident with the onset of endoreduplication, but the identity of this inhibitor is unknown. We therefore tried to determine if cyclin-dependent kinase inhibitor (CKI) activity is required for the occurrence of endoreduplication in maize endosperm. The expression of two maize CKI genes, Zeama;CKI;1 and Zeama;CKI;2, were characterized in developing endosperm. The accumulation of Zeama;CKI;1 RNA is not developmentally regulated, and its expression encompasses the period in which endoreduplication takes place. In contrast, Zeama;CKI;2 gene expression is developmentally regulated in the endosperm, since its protein level decreases after 13 DAP. Both proteins were able to inhibit the maize Cdc2/CDK kinase activity associated with p13 Suc1. They also specifically inhibited cyclin A1;3/ and cyclin D5;1/-associated CDK activities, but not cyclin B1;3/CDK. Although Zeama;CKI;1 was found to be associated with the endosperm CKI activity, it did not account for all of the CDK inhibitor. Over-expression of ZeamaCKI1 in maize embryonic calli that ectopically expressed the wheat dwarf virus RepA protein, which counteracts retinoblastoma-related protein (RBR) function in the cell cycle, led to an additional round of DNA replication without nuclear division. However, a role for Zeama;CKI;1 in endoreduplication could not be demonstrated in maize endosperm.

Withholding irrigation to alfalfa (Medicago sativa L.) during summer, a management strategy referred to as summer irrigation termination (SIT), has been suggested as a way to conserve water in desert environments. SIT may decrease productivity of alfalfa stands, although such negative effects may be reduced if cultivars with improved tolerance to SIT could be developed. This research was undertaken to determine how improved tolerance to SIT could be achieved through plant breeding. Single spaced plants of an extremely nondormant alfalfa population were grown in a field trial in Tucson, AZ and exposed to SIT in 1994 and 1995. These plants were used to identify traits associated with tolerance to SIT and represented parental material in a selection experiment. Direct selection for minimal reduction of forage yield following SIT was conducted under two stress intensities (lengths of SIT) and compared to indirect selection for characteristics potentially associated with dehydration avoidance. None of these selection criteria improved post-SIT forage yield relative to a random sample of plants from the parental population. This lack of response from selection was attributed to stress intensities that were not sufficiently high to fully expose genetic variation for yield following SIT. Physiological studies showed that high concentrations of total nonstructural carbohydrates (TNC) in crown tissue are positively associated with tolerance to SIT. Using TNC concentrations as an indirect selection criterion may therefore represent a more promising approach in improving tolerance to SIT than direct selection for post-SIT yield. Crown tissue was shown to die if the tissue moisture content fell below about 42%. This threshold value was used to predict whole-plant mortality of alfalfa grown in solid-seeded plots comparable to commercial fields. Crown samples were taken at five locations within the field along a soil gradient that caused whole-plant mortality to vary from 0.5 ± 0.5 to 48.7 ± 4.1%. Predicted values closely followed this change in observed mortality rates (r² = 0.97*) but tended to overestimate actual mortality on average by 4.2%. Alfalfa growers may be able to minimize mortality using this simple method to predict mortality during SIT and to reschedule irrigation accordingly.

Saguaro cactus virus (SCV) is a single-stranded RNA virus which belongs to the carmovirus genus within the family Tombusviridae. A full-length infectious clone of SCV has been generated and in this study was used to: (1) elucidate the role of the capsid protein (CP) in cell-to-cell and long distance movement, and (2) to better understand the various function(s) of the p26 and p86 proteins in viral replication. Analysis of a series of frameshift mutants and a deletion mutant has demonstrated that the CP is required for cell-to-cell movement in both Chenopodium amaranticolor and C. capitatum. This analysis also revealed a requirement of the CP coding region for viral replication in protoplasts. This is the first report of a cis-element, required for tombusvirus replication, which extends beyond the 3'-untranslated region into the CP coding region. The p26 and p86 constitute the putative SCV RNA-dependent RNA polymerase (RdRp). To better understand the structure and function of the RdRp, 16 clustered charged-to-alanine mutants were generated in the p26 and p86. The infectivity as well as the ability of each of these mutants to replicate in protoplasts was analyzed and compared to the infectivity and replication level of the wild type (pSCV15). Of the 16 mutants, five of them were nearly as infectious as wild type and were also able to replicate at near wild type levels. Four of the mutants consistently displayed a lower replication rate as determined by Northern analysis with two of these four demonstrating a lower level of infectivity on indicator plants. Two other mutants demonstrated a level of replication which was only able to be detected by RT-PCR. These mutants were not able to elicit the formation of local lesions on C. amaranticolor or induce symptoms on either inoculated or systemic leaves of C. capitatum. The ability of these mutants to synthesize negative-strand RNA, was examined. It was determined that all of the mutants which were able to produce positive-strand RNA were also able to synthesize negative strand RNA as determined by RT-PCR. Five of the mutants were not able to replicate in protoplasts and were not infectious on either host. These remaining five uninfectious mutants were also unable to replicate either negative or positive-strand RNA.

Many plant genes that are not expressed under normal growth conditions are activated in response to low temperature, drought, or salt stress. Plants must sense the stress they are under, then transmit the signal to the cellular machinery and activate stress-regulated genes. To help understand the signal events involved in the process, we used the firefly luciferase reporter gene driven by the stress-responsive RD29A promoter to screen for Arabidopsis mutants defective in stress signaling. In this study, the identification of several genetic loci is reported. Mutations in the FIERY1 locus resulted in increased gene expression under low temperature, drought, salt, and abscisic acid (ABA) treatments. FIERY1 thus underlies a connecting point of these diverse signaling pathways. FIERY1 encodes an inositol polyphosphate 1-phosphatase and is proposed to mediate the degradation of the second messenger inositol 1,4,5-trisphosphate. On the other hand, mutation in the SAD1 (s̲upersensitive to A̲BA and d̲rought 1) locus rendered the mutant plants more sensitive in gene expression, seed germination and seedling growth to ABA and salt/drought stress, but the response to cold was not changed. sad1 is also defective in drought-induced ABA biosynthesis and is impaired at the last step of ABA biosynthesis, i.e. the conversion of ABA aldehyde to ABA. SAD1 encodes an Sm-like U6 snRNP and is predicted to participate in mRNA processing. Two other loci defined in this study were found to encode enzymes in the ABA biosynthetic pathways. LOS5 encodes a molybdenum cofactor sulfurase and LOS6 encodes a zeaxanthin epoxidase. Mutations in these loci diminished osmotic stress-induced gene expression, suggesting that osmotic stress signaling does require ABA. Through studies with SAD1, LOS5 and LOS6 loci, a feedback regulatory loop was also identified. In this regulatory loop, ABA stimulates the expression of ABA biosynthetic genes, and this self-regulation may confer a rapid response to osmotic stress by speeding up ABA biosynthesis. These genetic, molecular, and biochemical studies provide many new insights into the signal transduction mechanisms in response to environmental stresses, and present successful examples of a molecular genetic approach to understand complex processes such as stress signal transduction.