Functional studies of lignin biosynthesis genes and putative flowering gene in Miscanthus x giganteus and studies on indolyl glucosinolate biosynthesis and translocation in Brassica oleracea

The first topic area of this thesis involved studies on the accumulation and translocation of glucosinolates (GSs), bioactive secondary plant compounds, in broccoli plants. Changes in GS accumulation and gene expression levels in response to exogeneous methyl jasmonate (MeJA) treatment were analyzed in different tissue types at different developmental stages of broccoli. Greater accumulation of GSs with MeJA treatment was observed in apical leaves of broccoli seedlings and florets of plants at harvest maturity. Increases in indolyl GS in apical leaves of seedlings and florets were coupled with the up-regulation of indolyl GS biosynthesis genes. The accumulation of indolyl GSs appears to be modulated by MeJA treatment in an organ-specific manner for optimal distribution of defense substances in the plant. Metabolic profiling of hydrophilic metabolites using GC-MS demonstrated increased accumulation of various phenolics, ascorbates and amino acids in broccoli tissues after MeJA treatment. Distinct changes in carbohydrate levels observed between different tissues (vegetative leaves and floret tissues) of broccoli plants after treatment suggest that carbon metabolism is differentially modulated by MeJA treatment in different tissue types depending on sink-source relationships. Reduced levels of hexose sugars and tricarboxylic acid intermediates after MeJA treatment may reflect the increased requirement for carbon and energy needed to drive secondary product biosynthesis to accumulate metabolites for defense against insects and other herbivores. Substantial increases of indolyl and aromatic GSs after exogenous treatment with MeJA in stem and petioles of seedlings and the existence of intact indolyl-GS forms in phloem exudates suggest enhanced de novo synthesis in combination with active transport. Indoly GSs share structural similarities with the auxin, IAA, and may interact with components of the auxin transport system for intra- and extra-cellular transport or translocation. Application of the auxin efflux inhibitor, 1-naphthylphthalamic acid (NPA) reduced MeJA-mediated accumulation of indolyl GSs in broccoli florets and seedling tissues. NPA did not inhibit expression of indolyl GS biosynthesis genes shown to be upregulated by MeJA treatment or the accumulation of tryptophan, the amino acid precursor of indolyl GSs. Exogenous application of benzyl GS to Arabidopsis roots induced ectopic expression of the PIN1 protein associated with the auxin transport system similar to treatment with NPA, again suggesting GS interaction with the auxin efflux carrier system. The inhibitory effect of NPA on MeJA-mediated accumulation of GS may be due to competitive binding of NPA to auxin efflux carrier components and that GS transport is mediated by the auxin transport system. The inhibitory effect of NPA on indolyl and aromatic GS accumulation and the bioactivity of exogenous treatment of these GS compounds in PIN1 localization, Arabidopsis root growth, and gravitrophic response suggest that indolyl and aromatic GSs may be antagonistic to IAA transport and biosynthesis. Indolyl and aromatic GSs can also be potentially converted into IAA by hydrolysis. This intrinsic feature of GSs may be the part of a sophisticated regulatory process where the metabolic pathways in the plant shift from active growth to a reversible defense posture in response to biotic or abiotic stress. It seems likely that indolyl and aromatic GSs are important compounds that provide connections between jasmonate and auxin signaling. Further studies are required to reveal the regulatory mechanism for crosstalk between the two hormones. The third part of this research was to investigate effect of selenium fertilization and MeJA treatment on accumulation of GSs in broccoli florets. Increasing dietary intake of the element selenium (Se) has been shown to reduce the risk of cancer. Simultaneous enhancement of both Se and GS concentrations in broccoli floret tissue were conducted through the combined treatment of MeJA with Se fertilization. A low level of Se fertilization (concentration) with MeJA treatment displayed no significant changes in total aliphatic GS concentrations with 90% and 50% increases in indolyl and total GSs concentrations, respectively. This result suggests that Se- and GS-enriched broccoli with improved health-promoting properties can be generated by this combined treatment. The second topic of this thesis was conducted to provide basic information required to improve biomass quality and productivity and develop tools for gene transformation in Miscanthus x giganteus. The perennial rhizomatous grass, Miscanthus x giganteus is an ideal biomass crop due to its rapid vegetative growth and high biomass yield potential. As a naturally occurring sterile hybrid, M. x giganteus must be propagated vegetatively by mechanicalling divided rhizomes or from micropropagated plantlets. The effect of callus type, age and culture methods on regeneration competence was studied to improve regeneration efficiency and shorten the period of tissue culture in M. x giganteus propagation. Seven lignin biosynthesis genes and one putative flowering gene were isolated from M. x giganteus by PCR reactions using maize othologous sequences. Southern hybridization and nuclear DNA content analysis indicated that the genes isolated from M. x giganteus exist in the genome of other Miscanthus species as multiple copies. Analysis of lignin content and histological staining of lignin deposition indicated that higher lignin content is found in mature stem node tissues compared to young leaves and apical stem nodal tissues. Cell wall lignification is associated with increasing tissue maturity in Miscanthus species. RNAi and antisense constructs harboring sequences of these genes were developed to generate Miscanthus transgenic plants with suppressed of lignin biosynthesis and delayed flowering.

Quichua-Spanish language contact in Salcedo, Ecuador: revisiting media lengua syncretic language practices

The purpose of the current thesis is to develop a better understanding of the interaction between Spanish and Quichua in the Salcedo region and provide more information for the processes that might have given rise to Media Lengua, a ‘mixed’ language comprised of a Quichua grammar and Spanish lexicon. Muysken attributes the formation of Media Lengua to relexification, ruling out any influence from other bilingual phenomena. I argue that the only characteristic that distinguishes Media Lengua from other language contact varieties in central Ecuador is the quantity of the overall Spanish borrowings and not the type of processes that might have been employed by Quichua speakers during the genesis of Media Lengua. The results from the Salcedo data that I have collected show how processes such as adlexification, code-mixing, and structural convergence produce Media Lengua-type sentences, evidence that supports an alternative analysis to Muysken’s relexification hypothesis. Overall, this dissertation is developed around four main objectives: (1) to describe the variation of Spanish loanwords within a bilingual community in Salcedo; (2) to analyze some of the prominent and recent structural changes in Quichua and Spanish; (3) to determine whether Spanish loanword use can be explained by the relationship consultants have with particular social categories; and (4) to analyze the consultants’ language ideologies toward syncretic uses of Spanish and Quichua. Overall, 58% of the content words, 39% of the basic vocabulary, and 50% of the subject pronouns in the Salcedo corpus were derived from Spanish. When compared to Muysken’s description of highlander Quichua in the 1970’s, Spanish loanwords have more than doubled in each category. The overall level of Spanish loanwords in Salcedo Quichua has grown to a level between highlander Quichua in the 1970’s and Media Lengua. Similar to Spanish’s lexical influence in Media Lengua, the increase of Spanish borrowings in today’s rural Quichua can be seen in non-basic and basic vocabularies as well as the subject pronoun system. Significantly, most of the growth has occurred through forms of adlexification i.e., doublets, well-established borrowings, and cultural borrowings, suggesting that ‘ordinary’ lexical borrowing is also capable of producing Media Lengua-type sentences. I approach the second objective by investigating two separate phenomena related to structural convergence. The first examines the complex verbal constructions that have developed in Quichua through Spanish loan translations while the second describes the type of Quichua particles that are attached to Spanish lexemes while speaking Spanish. The calquing of the complex verbal constructions from Spanish were employed when speaking standard Quichua. Since this standard form is typically used by language purists, I argue that their use of calques is a strategy of exploiting the full range of expression from Spanish without incorporating any of the Spanish lexemes which would give the appearance of ‘contamination’. The use of Quichua particles in local varieties of Spanish is a defining characteristic of Quichuacized Spanish, spoken most frequently by women and young children in the community. Although the use of Quichua particles was probably not the main catalyst engendering Media Lengua, I argue that its contribution as a source language to other ‘mixed’ varieties, such as Media Lengua, needs to be accounted for in descriptions of BML genesis. Contrary to Muysken’s representation of relatively ‘unmixed’ Spanish and Quichua as the two source languages of Media Lengua, I propose that local varieties of Spanish might have already been ‘mixed’ to a large degree before Media Lengua was created. The third objective attempts to draw a relationship between particular social variables and the use of Spanish loanwords. Whisker Boxplots and ANOVAs were used to determine which social group, if any, have been introducing new Spanish borrowings into the bilingual communities in Salcedo. Specifically, I controlled for age, education, native language, urban migration, and gender. The results indicate that none of the groups in each of the five social variables indicate higher or lower loanword use. The implication of these results are twofold: (a) when lexical borrowing occurs, it is immediately adopted as the community-wide norm and spoken by members from different backgrounds and generations, or (b) this level of Spanish borrowing (58%) is not a recent phenomenon. The fourth and final objective draws on my ethnographic research that addresses the attitudes of syncretic language use. I observed that Quichuacized Spanish and Hispanicized Quichua are highly stigmatized varieties spoken by the country’s most marginalized populations and families, yet within the community, syncretic ways of speaking are in fact the norm. It was shown that there exists a range of different linguistic definitions for ‘Chaupi Lengua’ and other syncretic language practices as well as many contrasting connotations, most of which were negative. One theme that emerged from the interviews was that speaking syncretic varieties of Quichua weakened the consultant’s claim to an indigenous identity. The linguistic and social data presented in this dissertation supports an alternative view to Muysken’s relexification hypothesis, one that has the advantage of operating with well-precedented linguistic processes and which is actually observable in the present-day Salcedo area. The results from the study on lexical borrowing are significant because they demonstrate how a dynamic bilingual speech community has gradually diversified their Quichua lexicon under intense pressure to shift toward Spanish. They also show that Hispanicized Quichua (Quichua with heavy lexical borrowing) clearly arose from adlexification and prolonged lexical borrowing, and is one of at least six identifiable speech styles found in Salcedo. These results challenge particular interpretations of language contact outcomes, such as, ones that depict sources languages as discrete and ‘unmixed.’ The bilingual continuum presented in this thesis shows on the one hand, the range of speech styles that are accessible to different speakers, and on the other hand, the overlapping, syncretic features that are shared among the different registers and language varieties. It was observed that syncretic speech styles in Salcedo are employed by different consultants in varied interactional contexts, and in turn, produce different evaluations by other fellow community members. In the current dissertation, I challenge the claim that relexification and Media Lengua-type sentences develop in isolation and without the influence of other bilingual phenomena. Based on Muysken's Media Lengua example sentences and the speech styles from the Salcedo corpus, I argue that Media Lengua may have arisen as an institutionalized variant of the highly mixed "middle ground" within the range of the Salcedo bilingual continuum discussed above. Such syncretic forms of Spanish and Quichua strongly resemble Media Lengua sentences in Muysken’s research, and therefore demonstrate how its development could have occurred through several different language contact processes and not only through relexification.