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It is now well established that cold-triggered calcium influx mediates cold-induced gene expression and development of freezing tolerance (cold acclimation). In this thesis, cold signaling events both upstream and downstream of calcium influx were examined. / First, it was shown that the studies on calcium mediation of cold acclimation in alfalfa cell suspension cultures could be applied to intact seedlings of Arabidopsis. Calcium chelators and channel blockers caused a strong reduction in the cold-induced accumulation of kin1 and kin2 transcripts, suggesting that calcium influx was an essential event during cold signaling and that the source of calcium for this influx was largely the calcium-rich cell wall. Evidence suggesting the involvement of calcium-dependent protein kinases (CDPKs) was also obtained. / Second, the nature of events upstream of calcium influx was explored. For this study, transgenic Brassica napus seedlings possessing both the endogenous cold-inducible BN115 gene and the coding part of beta-glucuronidase (GUS) gene placed under the control of the BN115 promoter were used. Thus cold-activation of the BN115 promoter drove the expression of both BN115 at the transcriptional level and the GUS enzyme activity at the translational level. Cold-activation of BN115 was inhibited by chemicals which cause membrane fluidization, cytoskeletal stabilization and inhibition of Ca2+ influx, and mimicked at 25°C by chemicals causing membrane rigidification, cytoskeletal destabilization and Ca2+ influx. Inhibitors of protein and lipid kinases prevented cold-activation of BN115, but inhibition of protein phosphatases activated BN115 at 25°C. / Third, given the increasing importance of mitogen-activated protein kinases (MAPKs) in signal transduction, the nature of molecular mechanisms that lead to cold-activation of a previously reported MAPK, SAMK, was investigated. During this study, the first plant MAPK activated by heat shock was discovered and named HAMK (Heat-shock-activated MAPK). It was shown that cold-activation of SAMK is mediated by cold-induced membrane rigidification, whereas the heat shock-activation of HAMK occurs through heat shock-induced membrane fluidization. Whereas activation of both SAMK and HAMK is blocked by an actin microfilament stabilizer, it is mimicked at 25°C by chemical destabilizers of microtubules or actin microfilaments. All of these events are inhibited by blocking the influx of extracellular Ca 2+. Cold-activation of SAMK and heat-activation of HAMK was prevented by treatment of cells with inhibitors of CDPKs. Thus, cold and heat shock are sensed by structural changes in the plasma membrane, which transduces the signal via cytoskeletal rearrangements to the opening of calcium channels, leading to Ca2+ influx, activation of CDPKs and activation of distinct MAPK cascades.

Nod factors, lipo-chitooligosaccharides (LCOs), are rhizobial signal molecules important in the establishment of nodule formation, leading to atmospheric dinitrogen fixation in legume-rhizobium symbioses. Recently, LCOs were also found to regulate other plant processes. We demonstrated that, at 10 -6 M, four LCOs produced by Bradyrhizobium japonicum enhanced soybean seed germination. Evaluation of G-protein inhibitors showed that U-73122, a phospholipase C inhibitor, also increased soybean seed germination, similar to the increase by LCO NodBj-V(C18:1 MeFuc), indicating different mechanisms for the plant perception to LCOs for nodule initiation and seed germination. This was confirmed as LCOs were not able to break dormancy of skotodormant lettuce seeds. Soybean early seedling growth was also increased by the application of LCOs. Pulse 14Ca2+ experimentation showed that the increase might also be related to an increase in Ca 2+ uptake by shoots. We confirmed this with both genistein-induced and non-induced B. japonicum 532C; however, strain 168 (a mutant unable to produce LCO) and non-host rhizobia (Rhizobium leguminosarum, Sinorhizobium meliloti), did not increase Ca2+ uptake. Addition of 1.6 g L-1 casein hydrolysate in yeast extract mannitol broth drastically increased bacterial growth and increased volume-basis LCO production, but decreased LCO production per cell. Best conditions for sterilizing and storing LCOs were determined. LCO should be sterilized by using polyestersulfone filter or autoclaving for up to 30 minutes. LCO was degraded faster when stored at room temperatures (23 +/- 2°C) than low temperature (4 +/- 1°C) and can be stored more than one year.

The yeast SKN7 gene encodes a transcription factor that is involved in a variety of processes in cell physiology including cell wall synthesis, cell cycle progression, and oxidative stress resistance. Using a transcriptional reporter-based system, it has been demonstrated that Skn7p is regulated by the two-component osmosensor Sln1p in a manner that requires the phosphorelay molecule Ypd1p, but not the response regulator Ssk1p. Consistent with its regulation by an osmosensor, Skn7p is involved in negative regulation of the osmoresponsive HOG MAP kinase cascade. Cells lacking SKN7 and the protein serine/threonine phosphatase encoded by PTC1 are severely disabled for growth, and hyperaccumulate intracellular glycerol. The growth defect of skn7Delta ptc1Delta mutants can be bypassed by overexpression of specific phosphatase genes, or by deletion of the HOG MAP kinase pathway-encoding genes PBS2 or HOG1. / MID2 was isolated in a screen designed to identify upstream regulators of Skn7p. Mid2p is an extensively O-mannosylated protein that is localized to the plasma membrane. Mutants with defective beta-1,6-glucan synthesis grow more quickly when MID2 is absent. Conversely, MID2 is essential for viability in cells lacking FKS1, the gene encoding the primary catalytic subunit of beta-1,3-glucan synthase. mid2Delta mutants are resistant to calcofluor white, a drug that interferes with cell wall chitin synthesis, while cells overexpressing MID2 are supersensitive to the drug. mid2Delta mutants have a significant reduction in stress-induced chitin synthesis, while cells overexpressing MID2 hyperaccumulate cell wall chitin. Consistent with a proposed role in sensing and responding to cell wall stress, high copy expression of specific components of the cell wall integrity MAP kinase cascade suppress various mid2Delta phenotypes, and Mid2p is essential for full activation of the Mpk1p MAP kinase during various cell wall stress and morphogenic conditions. / Observations from genetic and biochemical experiments suggest that Mid2p is a regulator of the small G-protein encoded by RHO1. Deletion of MID2 is lethal to mutants lacking the Rho1p GEF Rom2p, but suppresses the low temperature growth defect of mutants lacking the Rho1p GAP Sac7p. Conversely, high copy expression of MID2 is a strong suppressor of mutants lacking TOR2, an upstream activator of Rom2p, but is toxic to sac7Delta mutants. High copy expression of MID2 causes increased GEF activity towards Rho1p. Mid2p appears to act in parallel to Rom1p and Rom2p in promoting GDP-GTP exchange for Rho1p in a mechanism that is not yet understood.

With the projected increase in global population, unprecedented increases in crop production will be needed and legume crops are one of the primary means of achieving these increases. The legume-Rhizobium symbiosis is the single most important source of biologically fixed nitrogen in agricultural systems but, as a biological system it is complex, and very sensitive to environmental effects, such as available soil nitrogen, soil pH (both high and low), soil salinity and extremes of soil temperature. Each of these may affect the delicate signal exchange process that occurs during symbiosis establishment. To better understand the effect of environmental factors on signal exchange and nodulation, we conducted four experiments, under controlled-environment conditions, with pea (Pisum sativum) as the model legume. The first experiment studied the effects of available nitrogen, the second the effects of low soil pH, the third the effect of soil salinity and the fourth the effects of low soil temperature. In all experiments the plants were inoculated with Rhizobium leguminosarum bv. viceae cells previously treated with 10 x 10-3 M of hesperitin or naringenin, or not treated (control). In all experiments plants were destructively sampled at 10, 20, 30 and 40 days after inoculation (in two experiments samplings were also conducted at 15 DAI), and data on plant and nodule variables were collected. To allow for a better understanding of the effects of flavonoids on nodule development an image analysis technique was developed that permitted us to measure every individual nodule at each sampling. This provided a more precise picture of nodule development over time than would have been possible with previous methods. Our results indicated that addition of flavonoids had positive effects on nodulation, both in number and size of nodules produced, and that the positive effects were greatest at the most inhibitory levels of the environmental factors tested, and at earlier sampling

Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), collectively termed as jasmonates, are naturally occurring in plants and are important signal molecules involved in induced disease resistance and stress responses of plants. Besides their role in-planta, they are also rhizosecreted by root cells. Germinating soybean seeds exude large quantities of jasmonic acid; however there is no knowledge regarding how jasmonates influence cells of the soybean symbiont, Bradyrhizobium japonicum, in the rhizosphere. We studied the role of jasmonates in the soybean-Bradyrhizobium symbiosis. Bradyrhizobium japonicum cultures were induced with jasmonates and the resulting Nod factors were isolated and purified. Our results showed that JA and MeJA strongly induced the production of Nod factors by the tested B. japonicum strains. When added together, genistein and jasmonates resulted in greater LCO production than either one alone. Jasmonic acid is produced from linoleic and linolenic acids via the octadecanoid pathway; we studied the effect of these two fatty acids on B. japonicum nod gene induction. Interestingly both linoleic and linolenic acids induced the nod genes and caused LCO production by B. japonicum cultures. Since jasmonates induced the nod genes and also caused LCO production in B. japonicum, I conducted experiments in the greenhouse and field to determine whether incubation of B. japonicum with JA or MeJA prior to inoculation increases soybean nodulation variables and grain yield. Both genistein and McJA increased nodule number and nodule dry weight per plant. Due to enhanced nitrogen fixation, attributed to increased nodule number and weight, soybean dry matter accumulation and grain yield were increased. These results document the discovery of jasmonates and their precursors as new signal molecules in the Bradyrhizobium - soybean nitrogen fixing symbiosis.

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Thesis (Ph. D.)--State University of New York at Binghamton, Department of Biological Sciences, 2009. / Includes bibliographical references.