<p>Phosphorus is a macronutrient required for plant growth and reproduction. Insufficient supplies of phosphate will adversely impact plant growth. In an effort to supply adequate phosphate to crops, large quantities of phosphate-rich fertilizer are applied to fields but much of the phosphate can leach from the soil as run-off, impacting water systems. Therefore, proper management of phosphate and the development of phosphate efficient genotypes of plants are strategies needed for a sustainable agriculture industry.</p> <p>This thesis project focused on the development of biomarkers of phosphate stress in <em>Thellungiella salsuginea, </em>a plant highly tolerant to salt, cold and water deficit. Biomass determinations and real-time quantitative PCR were used to determine the gene expression of several genes selected as known phosphate-responsive genes from studies of phosphate starvation of the related genetic model plant <em>Arabidopsis thaliana.</em></p> <p><em> Thellungiella </em>seedlings were grown on 5 and 500 µM phosphate media. The expression of several genes (<em>RNS1, At4, Pht1;1, Pht1;4, Pht1;5, Siz1, PHR1, WRKY75, </em>and<em> Pht2;1</em>) were assayed for their response to media phosphate content. <em>RNS1</em> and <em>At4 </em>expression was estimated from cDNA prepared from shoot tissues while <em>At4, Pht1;1</em> and <em>Pht1;5</em> expression was determined from root tissues. In all tissue sources, significantly increased expression of <em>RNS1</em>, <em>At4</em>,<em> Pht1;1</em> and <em>Pht1;5</em> was observed under 5 µM phosphate exposure.</p> <p><em> </em>Two natural accessions of <em>Thellungiella</em> were used in this study with one originating from the Yukon Territory, Canada and the second from Shandong Province, China. Seedlings of both ecotypes were grown on defined media plates containing various concentrations of phosphate (0, 25, 125, 250, 500, and 2000 µM). For both accessions, the addition of as little as 25 µM phosphate led to significant increases in root and shoot biomass. Gene expression levels corresponding to <em>RNS1, At4</em> and <em>Pht1;1</em> were the highest in Yukon and Shandong <em>Thellungiella </em>grown on 0 µM phosphate media. The addition of 25 µM phosphate to the media was enough to significantly decrease transcript abundance of <em>RNS1, At4 </em>and <em>Pht1;1. </em>In a test using the transfer of Yukon <em>Thellungiella </em>seedlings from high (500 µM) to low (5 µM) phosphate the expression of <em>At4</em> in roots and shoots increased 30-fold over a five-day period and only <em>Pht1;1</em> expression increased in the roots over the same time period.</p> <p><em>RNS1</em> and <em>At4</em> share attributes that make them suitable biomarkers for phosphate stress in plants. Both genes are expressed in the shoots making it easier to remove tissue for monitoring gene expression, and both genes show readily discernible increases in transcript levels for determination by qPCR. At present, however, the role for their products in phosphate assimilation by plants is uncertain. This lack of knowledge is a deterrent to adopting these genes for widespread use as biomarkers. In particular, more work needs to be done to characterize factors that elicit their expression to test the specificity of their response to phosphate stress in <em>Thellungiella</em>.</p> / Master of Science (MSc)

<p>Light is a critical environmental signal that regulates every phase of the plant life cycle, from germination to floral initiation. Of the many light receptors in the model plant <italic>Arabidopsis thaliana</italic>, the red- and far-red light-sensing phytochromes (phys) are arguably the best studied, but the earliest events in the phy signaling pathway remain poorly understood. One of the earliest phy signaling events is the translocation of photoactivated phys from the cytoplasm to the nucleus, where they localize to subnuclear foci termed photobodies; in continuous light, photobody localization correlates closely with the light-dependent inhibition of embryonic stem growth. Despite a growing body of evidence supporting the biological significance of photobodies in light signaling, photobodies have also been shown to be dispensable for seedling growth inhibition in continuous light, so their physiological importance remains controversial; additionally, the molecular components that are required for phy localization to photobodies are largely unknown. The overall goal of my dissertation research was to gain insight into the early steps of phy signaling by further defining the role of photobodies in this process and identifying additional intragenic and extragenic requirements for phy localization to photobodies. </p><p>Even though the domain structure of phys has been extensively studied, not all of the intramolecular requirements for phy localization to photobodies are known. Previous studies have shown that the entire C-terminus of phys is both necessary and sufficient for their localization to photobodies. However, the importance of the individual subdomains of the C-terminus is still unclear. For example a truncation lacking part of the most C-terminal domain, the histidine kinase-related domain (HKRD), can still localize to small photobodies in the light and behaves like a weak allele. However, a point mutation within the HKRD renders the entire molecule completely inactive. To resolve this discrepancy, I explored the hypothesis that this point mutation might impair the dimerization of the HKRD; dimerization has been shown to occur via the C-terminus of phy and is required for more efficient signaling. I show that this point mutation impairs nuclear localization of phy as well as its subnuclear localization to photobodies. Additionally, yeast-two-hybrid analysis shows that the wild-type HKRD can homodimerize but that the HKRD containing the point mutation fails to dimerize with both itself and with wild-type HKRD. These results demonstrate that dimerization of the HKRD is required for both nuclear and photobody localization of phy.</p><p>Studies of seedlings grown in diurnal conditions show that photoactivated phy can persist into darkness to repress seedling growth; a seedling's growth rate is therefore fastest at the end of the night. To test the idea that photobodies could be involved in regulating seedling growth in the dark, I compared the growth of two transgenic Arabidopsis lines, one in which phy can localize to photobodies (<italic>PBG</italic>), and one in which it cannot (<italic>NGB</italic>). Despite these differences in photobody morphology, both lines are capable of transducing light signals and inhibiting seedling growth in continuous light. After the transition from red light to darkness, the PBG line was able to repress seedling growth, as well as the accumulation of the growth-promoting, light-labile transcription factor PHYTOCHROME INTERACTING FACTOR 3 (PIF3), for eighteen hours, and this correlated perfectly with the presence of photobodies. Reducing the amount of active phy by either reducing the light intensity or adding a phy-inactivating far-red pulse prior to darkness led to faster accumulation of PIF3 and earlier seedling growth. In contrast, the <italic>NGB</italic> line accumulated PIF3 even in the light, and seedling growth was only repressed for six hours; this behavior was similar in <italic>NGB</italic> regardless of the light treatment. These results suggest that photobodies are required for the degradation of PIF3 and for the prolonged stabilization of active phy in darkness. They also support the hypothesis that photobody localization of phys could serve as an instructive cue during the light-to-dark transition, thereby fine-tuning light-dependent responses in darkness.</p><p>In addition to determining an intragenic requirement for photobody localization and further exploring the significance of photobodies in phy signaling, I wanted to identify extragenic regulators of photobody localization. A recent study identified one such factor, HEMERA (HMR); <italic>hmr</italic> mutants do not form large photobodies, and they are tall and albino in the light. To identify other components in the HMR-mediated branch of the phy signaling pathway, I performed a forward genetic screen for suppressors of a weak <italic>hmr</italic> allele. Surprisingly, the first three mutants isolated from the screen were alleles of the same novel gene, <italic>SON OF HEMERA</italic> (<italic>SOH</italic>). The <italic>soh</italic> mutations rescue all of the phenotypes associated with the weak <italic>hmr</italic> allele, and they do so in an allele-specific manner, suggesting a direct interaction between SOH and HMR. Null <italic>soh</italic> alleles, which were isolated in an independent, tall, albino screen, are defective in photobody localization, demonstrating that SOH is an extragenic regulator of phy localization to photobodies that works in the same genetic pathway as HMR.</p><p>In this work, I show that dimerization of the HKRD is required for both the nuclear and photobody localization of phy. I also demonstrate a tight correlation between photobody localization and PIF3 degradation, further establishing the significance of photobodies in phy signaling. Finally, I identify a novel gene, <italic>SON OF HEMERA</italic>, whose product is necessary for phy localization to photobodies in the light, thereby isolating a new extragenic determinant of photobody localization. These results are among the first to focus exclusively on one of the earliest cellular responses to light - photobody localization of phys - and they promise to open up new avenues into the study of a poorly understood facet of the phy signaling pathway.</p> / Dissertation

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 (p. 144-156).

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Plant Biology." Includes bibliographical references (p. 131-142).

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

Plants have evolved a wide variety of physical and biochemical defense mechanisms to protect against herbivores and pathogens. When wounded, hybrid poplar (Populus trichocarpa X P. deltoides) upregulates a suite of defense-related genes, some of which encode anti-herbivore proteins. Among the most strongly insect- and wound- induced genes in poplar is an acid phosphatase gene (AP). APs are enzymes that function in hydrolyzing phosphate from P-monoesters and anhydrides and are involved in the remobilization of phosphate from these pools. However, APs may also play a role in the defense against insects by acting as anti-insect proteins. In poplar, AP mRNA induction occurs within 1.5 hours, which is similar to other known poplar defense genes. In the work described in this thesis, a 2 to 3-fold increase in the extractable AP activity was demonstrated in the leaves of saplings 4 days post wounding. These results suggest the poplar AP is part of the defense response against leaf-eating herbivores. In another type of defense reaction, when hybrid poplar is infected by the pathogen Melampsora medusae, which causes poplar leaf rust, flavonoid pathway genes are induced. This induction leads to the accumulation of proanthocyanidins (PAs), compounds with antimicrobial activity. The expression of several flavonoid-specific glycosyltransferase (UGTs) genes were correlated with these PA genes, suggesting a role for them in PA biosynthesis. Therefore, the second objective of this thesis was to functionally analyze these UGT genes. UGTs are enzymes which catalyze glycosylation reactions, which is typically one of the last steps in the biosynthesis of plant phenolic compounds. Active recombinant proteins for two highly induced poplar UGTs (PtUGT1 and PtUGT2) were generated, and sequence analysis grouped these proteins with others involved in the glycosylation of flavonols and anthocyanidins (UGT78 family), and not PA precursors as expected from microarray data. Enzymatic analysis of one of these proteins (PtUGT1) supports this phylogenetic grouping. By contrast, PtUGT2 does not use any known flavonoid substrates. To investigate the role of PtUGT1 in planta, transgenic poplars were produced that suppressed the expression of this gene using RNA interference. Phytochemical analysis of these knockdown plants were found to display decreased levels of PAs. Tissue survey analysis also implicates the PtUGT1 gene in PA biosynthesis since phytochemical analysis correlates with gene expression of PtUGT1 in the various tissues tested. Thus the data suggests that this UGT gene may be involved in PA biosynthesis. / Graduate

Thesis (M.S.)--Rutgers University, 2008. / "Graduate Program in Plant Biology." Includes bibliographical references (p. 28-31).

The ecology, species distribution, and population structure of Armillaria was investigated in the forests of Massachusetts. From 64 plots at 16 sites, 640 isolates of Armillaria were collected from six forest types (northern hardwoods, mixed oak, pitch pine, white pine, white pine/mixed oak, and eastern hemlock). Armillaria gallica proved to be the most abundant species, making up 316/640 (52%) of all isolations. This was followed by A. solidipes (219/640; 34%), A. mellea (46/640; 7%), A. calvescens (36/640; 6%), A. gemina (16/640; 3%), and A. sinapina (7/640; 1%). Armillaria gallica was routinely encountered causing significant decay of the lower bole on living hardwood hosts, especially oaks. The population structure of 153 isolates of A. gallica collected from mixed oak forests was investigated using amplified fragment length polymorphisms (AFLPs). From a total sampling area of 4.51 ha, 38 AFLP genotypes were discovered, yielding a figure of eight genets per hectare with the average A. gallica genet occupying 0.13 ha. When the effects of hydrolyzable tannins on in vitro growth were compared between A. calvescens and A. gallica, it was A. gallica that appeared better at oxidizing and metabolizing commercial tannins (tannic acid and gallic acid) along with black oak root bark extracts. This was determined through measurements of colony area and dry biomass, and suggests that A. gallica may be a better adapted pathogen of oak. In order to more accurately distinguish between isolates of A. calvescens and A. gallica, a three-gene phylogeny was reconstructed, using partial sequences of the elongation factor 1-alpha (tef1), RNA polymerase II (rpb2) and nuclear large subunit (nLSU) genes. After comparing 12 isolates each of A. calvescens and A. gallica that originated from across northeastern North America, only the tef1 gene could accurately distinguish these two species. Five single nucleotide polymorphisms were present between the two species and maximum likelihood and maximum parsimony methods grouped A. calvescens and A. gallica into monophyletic clades.

No description available.