Zhang, Xing-Hai

One methionine sulfoxide reductase A (TMSRA) and two methionine sulfoxide reductase
Bs (TMSRB 1 and TMSRB2) were isolated from tobacco plants. TMSRA showed
specificity for the reduction of Met-(S)-SO and both TMSRBs were specific for the
reduction of Met-(R)-SO. TMSRA was the cytosolic form and both TMSRBs were
plastid forms based on sequence comparison and expression tests. TMSRA and TMSRB2
could use either thioredoxin (TRX) or dithiothreitol (DTT) as reducing system, while
TMSRB 1 showed little activity with TRX but much more activity with DTT, which was
similar to the mitochondrial MSRB2 from mammals. Ferredoxin (FD) is not the reducing
system for Msrs, but might reflect the redox status in the cell and control the activity of
Msrs indirectly.

Understanding the genetic regulation of the response to wounding and wound
healing in fruiting plants is imperative to maintaining agricultural sustainability,
preserving the quality of food supplies, and ensuring the economic viability of
agriculture. Many genes are known to be induced by wounding, providing both structural
repair and defense. The KED gene in tobacco (Nicotiana tabacum) has been shown to be
induced by wounding. We have identified its homologue gene in tomato (Solanum
lycopersicum) that we named SlKED. We have analyzed gene expression pattern of
SlKED through tomato growth and development and in response to wounding as well as
hormonal and inhibitor treatments. We found that the plant hormone ethylene played a
major role in the expression of SlKED. To further identify evidence for physiological and
transductional functions of KED and SlKED, the tobacco KED gene was introduced to
tomato and overexpressed by the fruit tissue-active PUN1 promoter from pepper
(Capsicum annuum,). The expression of this gene was compared to the expression of the native SlKED gene and other known wound response genes in both the wild-type and
transgenic tomato plants. The upregulation of the native SlKED gene by wounding was
significantly muted in the tobacco KED-expressing transgenic plants. The expression of
other genes known to be associated with wound response transduction pathways was also
altered. Our studies implicate the KED gene in defense mechanisms for mechanical stress
in tomato plants.

Water scarcity induced by drought, temperature, and salinity has plagued agricultural sustainability in recent years with unprecedented revenue losses, raising concerns for worldwide food security. Recent studies have revealed unique botanical response mechanisms to combat water related stress, namely the expression of proteins known as the dehydrins. Dehydrin proteins have been shown to serve various intracellular protective functions. The gene for a SK5 type dehydrin from the arctic plant Cerastium arcticum (CaDHN) was introduced into tobacco plants and water deficit tolerance was evaluated. Plants overexpressing CaDHN displayed improved tolerance to salt stress, but no improvement was observed under drought stress.

The NRT2 (high affinity nitrate transporter 2) family is a part of the iHATS (inducible high affinity system) that studies have shown is responsible for the influx of nitrate into the plant cell after provision of nitrate. The ZmNRT2.1 from Zea mays was constitutively expressed in Nicotiana tabacum. To assess how over-expression of this foreign NRT2.1 affects nitrate influx by plants, nitrate content in leaf and root tissue, gene expression, and vegetal growth were analyzed in media with deficient or high nitrate concentrations (0.1, 1, or 10 mM). Compared to wild type plants: the transgenic lines had a significantly larger fresh weight in all nitrate conditions; primary root length was significantly longer in the 0.1 and 1 mM nitrate conditions; both the fresh weight and the primary root length were significantly higher when 50 mM NaCl was applied as a stress factor to medias containing 0.1 and 10 mM nitrate.

The β-glucuronidase (GUS) gene was isolated in 1986 from the bacterium Escherichia coli. Since then it has been widely used as a reporter gene in genetically modified organisms serving to study gene expression and tissue specificity of different promoter sequences. We have introduced the GUS gene into tobacco plants through Agrobacterium-mediated genomic transformation. The plants that were confirmed to be expressing the GUS gene were grown to propagate a new (T1) generation. The T1 plants were analyzed for tissue specificity of GUS expression. The results to date seem to indicate that there is some variation in GUS expression between plant lines. The mechanisms of GUS gene integration in the plant genome as well as the possible effects it can have on a plant’s genomic structure are assessed.

This thesis is intended to explore the genetic variation between cattail species (Typha spp.), within T. domingensis in different locations, anthropoegenic conditions, and possibly discover a hybrid in the Florida Everglades. Typha domingensis is the dominant cattail species in the Everglades, while Typha latifolia a less common species is also present. Five nuclear and chloroplast protein encoding genes from around 20 samples of cattail plants were collected randomly in the Water Conservation Areas of the Everglades Protection Act, cloned and sequenced. The results of sequencing showed differences between the two species studied, using an insertion within an intron of the Type 2 Metallothionein-like protein gene as a marker to differentiate between the two species. A high degree of nucleotide polymorphisms interspecifically was revealed. Species identification based on morphology is not always reliable that is why our marker must be utilized to confirm the identity of a plant.