Studies on plant nitric oxide synthase

Pao Yue-kong Library Electronic Theses Database

Studies on plant nitric oxide synthase


Author: Butt, Kwok-chu
Title: Studies on plant nitric oxide synthase
Degree: M.Phil.
Year: 2003
Subject: Hong Kong Polytechnic University -- Dissertations.
Nitric oxide.
Botanical chemistry.
Department: Dept. of Applied Biology and Chemical Technology
Pages: xxv, 268, [8] leaves, [12] p. : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record:
Abstract: Nitric oxide (NO) is identified as an important signaling molecule in plant growth, development and defense responses against pathogens. However, in plants, the source of endogenous NO is still controversial. Although neither a nitric oxide synthase (NOS)-like protein nor a gene has been found, many studies have suggested the existence of a mammalian-like NOS regarding the synthesis of NO in plants. As this NOS-like enzyme is crucial in understanding NO metabolism in plants, the aim of this study is to investigate whether this plant enzyme exists. Recently, several studies found immunoreactive proteins in plant homogenates by probing with either mammalian nNOS or iNOS antibodies. Therefore, in preliminary studies, a dot-blot system was used with the mammalian NOS antibodies to screen for the presence of NOS in various leguminous plants. Only one plant tested, the asparagus bean (Vigna sesquipedalis) showed a positive immunoreactivity, whereas no corresponding NOS activity could be found in its homogenate by a chemiluminescence assay. In addition, in our hands, a strong nNOS-immunoreactive band with an apparent molecular mass of 110kDa was strongly detected in embryonic axes of maize (Zea mays). This immunoreactive band has been ascribed to a plant NOS without any further identification in previous studies by two research groups. However, using N-terminal amino acid sequencing, it was identified as alpha-1',4'-glucan phosphorylase. These findings led us to verify the identities of plant proteins that cross-react with the mammalian NOS antibodies. Using tryptic digestion and MALDI-TOF mass spectrometry, many NOS-immunoreactive plant proteins were identified as glycolytic enzymes or heat shock proteins. No NOS-like enzyme could be found or identified. These results suggest that this immunological approach should not be used for plant NOS identification. As NOS cross-reactivity cannot be found, the possibility that NO production might be induced, was examined. Many recent studies reported that both NO and hydrogen peroxide (H2O2) are involved in plant defense responses. H202 is an important early signaling molecule that rapidly produces in plants following exposure to a variety of biotic and abiotic stimuli. It is thought that upon this oxidation challenge, NO could also be generated by a NOS-like enzyme in the defense responses. Therefore, mung bean (Phaseolus aureus) leaves were treated with H202 and a subsequent presence of NO was monitored by a NO specific fluorescent dye, DAF-2DA. A rapid NO production was observed after the H202 addition, and this production was found to be calcium dependent and inhibit by NG-nitro-L-arginine-methyl ester (L-NAME), a known mammalian NOS inhibitor. Moreover, a BH4-dependent NOS activity was also detected in the total protein extract from the H202 treated mung bean leaves. Besides, the existence of NOS enzyme was also examined in normal plant physiological processes, such as germination. During mung bean germination, a transient increase in a mammalian-like NOS activity was observed by the chemiluminence assay in its embryonic axes followed by an increase in cGMP level. Furthermore, purification of this putative plant NOS was performed. Protein extract from the embryonic axes showing NOS activity was fractionated by different column chromatographic techniques. Protein fractions eluted at around 0.5M NaCl in a DEAE Sepharose column was found to exhibit a NOS activity. However, the shelf-life of this NOS activity was found to be less than 24 hrs at 4 C. ln conclusion, results obtained so far suggested the presence of mammalian-like NOS in plants including maize and mung bean, either in leaves after H202 induction or in embryonic axes during germination. Importantly, together with the results from western blot analysis, this plant NOS should be a protein that is functionally similar, but structurally different from mammalian NOS species.

Files in this item

Files Size Format
b16927023.pdf 14.05Mb PDF
Copyright Undertaking
As a bona fide Library user, I declare that:
  1. I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
  2. I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
  3. I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.
By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.


Quick Search


More Information