Since the production of every enzyme along with its location and function within the cells of a given plant are ultimately controlled by the sequence of nucleotides on strands of DNA, one last category of factors that influence metabolite biosynthesis will be considered. These factors interact with the DNA molecules themselves to regulate the activity of the genes that govern the individual enzymes of each pathway.
The primary steps in gene expression are depicted in Figure 3.8. The steps of gene expression that take place in the nucleus to produce messenger RNA (DNA ^ Gene ^ Primary DNA Transcript ^ Mature mRNA) are known as tran-
scription. The steps of gene expression which take place in the cytoplasm to form polypeptide chains from this mRNA (mRNA e on the ribosomes ^ synthesis of polypeptides ^ formation of functional protein) are called translation. Note, in Figure 3.8, that l degradation of DNA, mRNA, and functional proteins can also occur when the appropriate hydrolases are present (DNAases, o RNAases, or proteases). Both synthesis and degradation of DNA, mRNA, and functional proteins are very important processes in gene regulation and are known as turnover. When the rate of synthesis exceeds the rate of degradation, there is a net synthesis m of DNA, RNA, or protein; when the converse occurs, there is a net loss of DNA, RNA, or protein. This has a direct impact on the t amount of production of a given enzyme within a given pathway. Synthesized mRNA and protein can also be stored within the o cells for later times when changing environmental conditions trigger their activation (e.g., long-lived mRNA in seeds and animals =e egg; storage proteins in seeds). The absolute level of DNA, mRNA, or a given protein in a cell or tissue will then depend rates at which synthesis, degradation, and storage take place. We refer to this system of regulation of the steady-state level of such h metabolites in cells as homeostasis. i
3.4.2 How Plant Genes Are Turned On and Off i a t
Regulation of gene expression can occur at the level of transcription (DNA to RNA), post transcription (initial RNA transcript s to mRNA, translation of mRNA to polypeptide), or post translation (polypeptide to functional protein). These levels of regulation are controlled by a range of environmental or developmental signals. The various control points in gene expression at these different levels are designated in Figure 3.8.
What are some of the developmental and environmental signals which regulate gene expression in plants? One is light that may up-regulate the synthesis of mRNA for synthesis of the light-harvesting complex involved in photosynthesis. This is mediated by the phytochrome system involving red and far-red wavelengths of light. Other signals are stresses elicited by such factors as ultraviolet light, wounding, or pathogen attack, which can up-regulate, at the level of transcription, the synthesis of such enzymes as PAL that leads to synthesis of phenylpropanoid compounds. Still other signals can be attributed to plant hormones which are bound by protein transcription factors within the cell. For example, in germinating cereal grass seeds, gibberellins (GAs) can cause de novo synthesis of mRNAs for a-amylase that break down starch to sugar and of proteases that can break down stored proteins in seeds. In contrast, the plant hormone, abscisic acid (ABA) turns off such gene expression in germinating seeds and is thus partly responsible for the dormancy of these seeds as well as the dormancy of the buds of temperate zone trees.
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The precise mechanisms by which environmental or developmental signals act to control gene expression are not yet completely understood. But, research so far has allowed several mechanisms to be promulgated including the following:
• The signal (such as GA or ABA) could stimulate the synthesis of a protein regulatory factor that binds to particular trans-acting (other proteins) or cis-acting (DNA sequence) elements located upstream in the promoter region of a gene to turn the gene on (as in the case of GA) or off (as in the case of ABA) gene expression.
• The signal (such as a cytokinin plant hormone, that acts to stimulate red light-induced synthesis of RuP2-Case and light-harvesting complex (LHCP) in greening tissue of duckweed, Lemna gibba) may act to stabilize particular mRNA species, i.e., retard the degradation of the initial RNA transcripts or mRNA produced from a given gene.9
• The signal may fail to act when plants are genetically engineered using constructs that have the gene of interest in anti-sense or reverse orientation. In this case the signal may be the plant hormone ethylene
that causes ripening in fruits due to enhanced activities of pectinases (a class of cell wall-loosening enzymes, more properly known as polyg-alacturonases, which hydrolyze pectins or polygalacturonans, the cementing substances located mostly in the middle lamella between primary cell walls). This has the effect of producing RNA molecules that are complementary to the normal (correct orientation) RNA. Since mRNA is single stranded, when these two molecules bind together through their mutual affinity, the normal mRNA will not function in translation. The FLAVR SAVR™ tomato is one such genetically engineered product where the gene for pectinase was introduced into tomato plants in an anti-sense orientation to knock out gene expression of the plant's pectinase.20
To produce such transgenic plants as the FLAVR SAVR™ tomato, there is a specific order of questions and answers that must be elucidated. In natural products research, one of the first important biochemical questions to ask is: "How is the metabolite of interest synthesized?" Another is: "What are the enzymes for the respective steps in the pathway?" These are not easy questions to answer, but once these enzymes are isolated and purified, then the molecular biologist can potentially clone the genes that make these enzymes, determine their nucleotide sequences, and characterize their expression patterns within the various plant tissues. At this point the pathway for the metabolite of interest will be well understood and a new question arises. How can the expression of the gene(s) for the rate-limiting enzyme(s) in the biosynthetic pathway be up-regulated, or down-regulated, so as to make more, or less, of the metabolite through genetic engineering protocols? These protocols include the use of constitutive or super promoters attached upstream of the gene, the use of constructs to make anti-sense RNA, and the use of genetic transformation to express the gene of interest in organisms that normally do not express this gene. If all of the biochemistry is done properly, including (a) the purification of the proteins of interest, (b) the characterization of any isozymes for the particular enzyme being studied as well as their ultimate site(s) of action in the cell, and (c) the elucidation of the function of the enzymes in cell metabolism, then the above-outlined molecular biology work is not only feasible, but also allows one to turn specific genes on or off in a particular metabolic pathway thus changing the production of specific metabolites. In doing this kind of work, risk assessments are absolutely necessary to determine if a particular transgenic plant can have any detrimental effect on our environment. These are discussed in detail in References 20 through 22.
3.4.3 Case Study: Isolation of Genes in the Isoprenoid Biosynthetic Pathway
Figure 3.9 illustrates the pathways of isoprenoid (also called terpenoid) biosynthesis in plants,23 and Table 3.5 provides a key to the enzymes that operate at each of the respective numbered steps in these pathways.23 The genes which have been cloned for many of the enzymes in the isoprenoid biosynthetic pathways are indicated in Table 3.6.23 Often natural product researchers will search the Genebank database for these cloned genes to obtain the nucleotide sequences of such genes (see Reference 24 on how to access the Genebank database). The information obtained can then be used to tackle the problem of increasing or decreasing the production of a specific metabolite within the plant. Let us take as an example the synthesis of natural rubber, which comes from plants such as the Brasilian rubber tree (Hevea brasiliensis) and guayule (Parthenium argen-tatum). This example focuses on the following question: How can one increase the levels of natural rubber in these plants? According to Dr. Katrina Cornish25 and C. Potena,26 natural rubber is made up of isoprene units that are derived from isopentenyl pyrophosphate (see Figure 3.9). The polymerization step is catalyzed by the enzyme, rubber transferase, which requires allylic pyrophosphate to initiate the process. Dr. Cornish has focused her attention on identifying, isolating, and manipulating rubber transferase and its two substrates, iso-pentenyl pyrophosphate and allylic pyrophosphate. Her results to date suggest that by raising the level of the initiator (through up-regulated gene expression of the enzyme which makes allylic pyrophosphate), she can enhance rubber production up to six times.26
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