The short-term effects of manganese toxicity on ribulose 1,5 bisphosphate carboxylase (Rubisco) activity and concentration in tobacco chloroplasts were examined. The activity of the enzyme from both manganese-treated and control plants was determined 6, 12, 18, 24 , and 48 h after introduction of manganese (80 mg/Li. Enzyme activity was determined by monitoring rates of radioactive CO2 fixation into acid stable products. A slight stimulation of the enzyme's activity was noted in experimental plants after 18 h of exposure to manganese as compared with control plants. A decrease in the enzyme's activity in experimental plants was noted after 48 h of exposure. Visible symptoms such as chlorosis and decreased leaf size were also observed after 48 h of manganese exposure in experimental plants. Using Rocket Immunoelectrophoresis, no appreciable difference between Rubisco concentration levels of the experimental plants and the control plants was noted after 6, 12, 18, 24, and 48 h of manganese exposure indicating that the effect on Rubisco activity is a post-translational phenomenon and that Rubisco is not being degraded at an accelerated rate. Even after 7 d of exposure to high manganese concentrations, when visible symptoms such as chlorosis and necrotic lesions were very evident, the level of Rubisco in the manganese-treated plants varied little from the levels in the control plants. Manganese accumulated in the experimental plants to concentrations as high as 3282 mg,/g dry wt as determined by atomic absorption spectrophotometry. A shuttling mechanism for manganese between young and old leaves was indicated by an observed decrease in the concentration of manganese in the young leaf tissue between 12 and 18 h after treatment .

The purposes of this study were to determine if Rhododendron macrophyllum has the potential to inhibit the growth of other species through the production of water-soluble toxins which are leached out of its litter by rainfall, and to determine if this potential is realized in the field. The study was therefore composed of two part: 1) a series of bioassays to determine the presence an activity of water-soluble phytotoxins in R. macrophyllum leaf litter, and 2) a field study to describe vegetational patterns associated with R. macrophyllum in a western Cascades clearcut.

A series of experiments evaluated: 1) the influence of seed intake and gut retention time on seed passage , recovery , and germinability; 2) fecal seeding and broadcast /trampling as techniques to incorporate seeds into a well-established Agropyron desertorum (Fisch.) Schult. stand in Skull Valley , Utah; 3) intensive grazing as a means to reduce Agropyron biomass and increase establishment and survival of seeded species; and 4) the recovery and germinability of seed extracted from dung collected from the field. Two shrubs (Artemisia tridentata Nutt. ssp. wyomingensis Beetle & Young and Atriplex confertifolia Torr. & Frem.), a grass (Elymus elymoides (Raf.) Swezey) , and a forb (Sphaeralcea grossulariaefolia (H. & A.) Rydb.) were selected as representative native species. Holstein heifers were fed 15,000 , 30 ,000 , and 60,000 seeds of Artemisia , Sphaeralcea, and Elymus. Elymus recovery was negatively correlated to seed intake. Sphaeralcea had the highest percentage of recovered, undamaged seed, followed by Elymus and Artemisia. Sphaeralcea and Artemisia seed passage was highest on Day 1 then dropped sharply. Elymus passage and recovery were more consistent through time. Post-passage germjnability was highest for Elymus and Sphaeralcea on Day 1. Artemisia germjnation was neghgible. In the fall seeding, Sphaeralcea emerged in 6% of the subplots (half were volunteers). Overall seedling mortality was 93%. Elymus emerged in 63% of the dung pats, with 86% mortality. No Artemisia emerged. Drought and Anabrus simplex herbivory contributed to low seedling emergence and survival. In April 2003, similar treatments were applied, except Atriplex seed was substituted for Artemisia, and a third treatment was added (broadcast seeding/raking). No emergence was observed. Sphaeralcea had the highest seed recovery from dung collected in the field trials, followed by Elymus, Atriplex, and Artemisia. Sphaeralcea germinability was similar for seeds collected from both trials (11 %) and Elymus germination was highest in the fall seeding (13%). These studies indicate that: 1) physical seed properties (size, shape, density, seed coat) influence seed passage , recovery, and germination; 2) intensive grazing can reduce Agropyron biomass by 50% for 2 years; 3) broadcast/trampling may be effective for Sphaeralcea; and 4) an average-sized dung pat (714 g) may have ample germinable Sphaeralcea and Elymus seeds for plant establishment.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Plant Biology." Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Plant Biology." Includes bibliographical references.

The ability to maintain hydraulic continuity in the xylem is essential to supply leaves with the water that must be exchanged for carbon dioxide. The metastable nature of xylem sap causes this system to be inherently vulnerable to failure by rapid vaporization within the conduits. Much of the recent work on hydraulic architecture and cavitation has pursued the elusive mechanism behind apparent hydraulic recovery concurrent with tension in the bulk of the xylem, referred to as "novel refilling". An investigation into the dynamics of this behavior (Chapter 3) revealed two key artifacts that can produce the appearance of novel refilling when in fact no embolism (and therefore, no recovery) has occurred. A further implication of these artifacts is that plant xylem may be more robust against embolism than previously expected. In the absence of novel refilling, it becomes much harder to reconcile the extreme vulnerability reported for ring porous species. Studies of Robinia pseudoacacia (Chapter 4) address whether the artifacts illuminated in chapter 3 provide insight into the ongoing debate about the cavitation resistance of long-vesseled species and whether it is possible to accurately assess cavitation resistance in these species using the centrifuge method. Root pressure, as an alternative to novel refilling, provides plants with a means of reversing cavitation. Studies of Betula papyrifera (Chapter 5), however, show that recovery from embolism by root pressure is limited to early spring and point to an important role for water storage in fibers that minimizes xylem tensions and thus the risk of cavitation.

Programmed cell death (PCD) is a fundamentally important process delicately coordinated throughout an organism’s life cycle. In plants, PCD is an integral part of development, reproduction, and pathogenesis. Numerous types of proteins are involved in regulation of PCD in plants, like phosphatases, metacaspases, and protein kinases. In tomato resistance to the pathogen Pseudomonas syringae pv. tomato (Pst), a Ser/Thr protein kinase, Adi3 (AvrPto-dependent Pto-interacting protein 3), interacts with the pathogen’s avirulence protein AvrPto and the tomato resistance protein Pto. Adi3 is a member of the AGC protein kinase family, a group known to transmit signals via the secondary messengers cAMP, cGMP, and phospholipids. In an unchallenged system, the master regulator of AGC kinases, Pdk1, activates Adi3. Activation of Adi3 enables nuclear localization and cell death suppression – all of which is prevented when challenged by Pst. A BLAST_p search of the Arabidopsis thaliania genome with the amino acid sequence of Adi3 identified a 67% identical match, AGC1-3. Like Adi3, AGC1-3 at its activation-loop serine and another site, by Pdk1. With N-terminal deletions of AGC1-3, Pdk1 was found to phosphorylate AGC1-3 at two serines – one serine conserved among all Arabidopsis AGC kinases, the other a serine on the N-terminus of the kinase domain. The non-activation loop serine in AGC1-3, Ser269, is conserved at Ser212, in Adi3. Phosphorylation at Ser212 does not impact auto-catalytic activity of Adi3. However it does enhance trans-catalytic activity. Analysis of AGC1-4 and AGC1-7, two proteins closely related to AGC1-3, reveals that phosphorylation of non-activation loop residues by Pdk1 is not restricted to AGC1-3 and Adi3. Functional analysis of AGC1-3 in Arabidopsis protoplasts revealed that like Adi3, nuclear localization and activation-loop phosphorylation are essential for cell viability. In an effort to elucidate a signaling network controlled by AGC1-3, the KiC (Kinase-client) assay was employed. In the KiC assay, a 2,100-member peptide library was assayed against AGC1-3 and the constitutively active mutant AGC1-3^(S596D). By MS analysis, AGC1-3 and AGC1-3(S596D) phosphorylated 26 and 19 substrate peptides, respectively. Substrate peptides were mapped to proteins involved in central metabolism, transcription, and protein metabolism. The work presented in this dissertation provides conclusive evidence that Pdk1 phosphorylates AGC1-3 and Adi3 at a non-activation loop residue. The work also supports AGC1-3 as the Arabidopsis homologue of Adi3 and presents novel phosphorylation data of potential AGC1-3 substrates.

<p>Roots are developmentally plastic and highly dependent on the immediate environment. By studying root responses to abiotic stress, we have identified novel regulators of developmental modulation. When roots are deprived of phosphate (Pi), developmental programs are modulated to slow primary root growth and expand surface area through emergence of root hairs. By focusing on exposure time-periods of less than two days, we have described very early changes to root development in response to this condition that may reveal new mechanisms of root hair specification and emergence. Also, using transcriptomic analyses with high spatial resolution, we identified a kinase that is specifically induced in root vascular tissue within three hours of exposure and acts to modulate aspects of root development in response to deprivation of Pi. These data suggest that individual tissues play unique roles in whole organ development, and that interpretation of Pi -deprivation responses may change as we develop methods with resolution necessary to understand these roles. Beyond Pi, we compared transcriptomic data for four additional stresses and identified a novel stress-responsive transcription factor that modulates expression of a cell expansion protein. This putative network connection demonstrates the value of using high-dimensional data for inference of regulatory relationships. Overall, we have combined "-omics" approaches with reverse genetics to identify novel developmental regulators and described a phenotypic frame-work with resolution at which cellular mechanisms can be studied.</p> / Dissertation

Transgenic lines of sugarcane with increased total sugar content have been obtained in previous work in our lab, through the introduction of a sucrose isomerase (SI) gene designed vacuole-targeting of the SI gene product. Some of the resulting transgenic lines accumulate the high value sugar isomaltulose (IM) without decrease in stored sucrose content. Other lines show enhanced sucrose content with low levels of IM in mature storage tissues. Under containment glasshouse conditions, these “SugarBooster™” lines have shown up to two-fold increase in the total sugar concentration in the harvested juice. This remarkable step above the former ceiling in stored sugar concentration may permit new insights into the mechanisms by which plants regulate sugar accumulation, a pivotal question in plant biology. Studies conducted in this thesis are a part of the effort to understand the underlying mechanisms. Experiments were focused on the regulation of SI and endogenous genes related to sucrose metabolism, as revealed by transcript levels. Enzyme activity was also tested for sucrose phosphate synthase (SPS), a key enzyme in plant sucrose biosynthesis. Initially, the relationship between IM content and SI transcript level was analyzed. Real-time quantitative polymerase chain reaction (RT-qPCR) successfully detected SI gene transcripts in internodes of different maturities in SugarBooster™ lines, but no correlation was found between the transcript level and IM content. In contrast, northern blotting to distinguish full-length SI mRNA from degraded transcripts revealed a positive relationship between IM content and functional transcripts of SI. The results imply that stabilization of SI mRNA is important for high IM content but that high sucrose accumulation accompanied by low IM involves other mechanisms. Therefore, transcript levels of several key genes in sucrose metabolism were analyzed in internodes at various developmental stages in SugarBooster™ iv and control lines. These genes encode sucrose phosphate synthase (SPS), sucrose synthase (SuSy), soluble acid invertase (SAI), neutral invertase (NI), cell wall invertase (CWI), a putative sugar transporter type 2a (Type2a) and sucrose transporter type 6 (Type6). Sucrose content was negatively correlated to the transcript levels of SAI, but there were no significant correlations between sugar content and mRNA levels of other tested enzymes across all developmental stages. However, interesting transcript patterns that might contribute to high sucrose accumulation were observed in some lines. Examples from different lines include: SuSy transcripts increased in immature internodes but decreased in mature ones; NI expression decreased in mature internodes and sugar transporters increased in some cases. Further analysis was undertaken on transcript regulation of five SPS families, as SPS is the rate-limiting enzyme for sucrose biosynthesis in some plant systems. As recently reported from a CSIRO analysis of sugarcane progeny segregating for sucrose content, transcripts of SPS family 2 predominated in the stem for both SugarBooster™ and control lines. Transcripts of SPS families 3 and 4 were less abundant and families 1 and 5 were rare. More abundant mRNAs of SPS families 2, 3 and 4 indicate that they play an important role in sucrose accumulation in the stem. However, none of the mRNA levels of the five families had a significant correlation to the SPS enzyme activity or to the sucrose content measured in corresponding tissues. In contrast, SPS enzyme activity was found positively correlated to sucrose content. It revealed that regulation of SPS in sugarcane is mainly at the level of enzyme activity. These studies have been constrained by the availability of experimental material in early vegetative generations of SugarBooster™ lines under containment glasshouse conditions. As the opportunity emerges for more material from approved field trials, it will be important to identify the most stable lines under field conditions, for further studies on metabolites, carbon partitioning and enzyme activity to fill the gap in understanding between regulation at transcript level and the SugarBooster™ phenotypes.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Plant Biology." Includes bibliographical references (p. 178-184).