Transgenic plants have particularly been found useful in the study of development, since with their help gene expression can be studied in time and space i.e. in different environmental conditions causing induction or suppression of gene expression. Transgenic plants thus facilitated the recognition of regulatory sequences involved in these differential expressions of gene activity. For this purpose, sequences upstream to a number of structural genes were cloned either with their own structural genes or with other unrelated structural genes (e.g. reporter genes)
to study the specific regulatory role of these sequences in time and space. A number of structural genes, whose regulatory sequences were examined in this manner are listed in Table 37.4, and the details of some of these are described in the following text.
Gene for small subunit of ribulose bisphosphate carboxylase (rbcS).
Dozens of genes in plants are affected by light and many of these regulated by phytochrome.
The expression of phytochrome gene is depressed and that of RUBISCO
(ribulose, 1,5 bisphosphate carboxylase/ oxygenase) gene is stimulated by red light. RUBISCO is involved in CO2
fixation and is the most abundant enzyme on the earth. It consists of two subunits, each encoded by a separate gene, the smaller subunit rbcS
encoded by a nuclear gene and the larger subunit rbcL
encoded by a chloroplast gene. Although, rbcS from different plants are fairly similar, small differences, that exist, can be used to distinguish mRNA of a transgene from that of the host plant using specific probes. The pea rbcS
gene was transferred to petunia and tobacco plants and soybean rbcS
gene was transferred to petunia. The gene expressed itself in transgenic plants, rbcS subunit from the introduced gene sometimes even combining with rbcL subunit of the host plant to yield the enzyme. 'Deletion mutants'
having sequences deleted upstream of the transgene rbcS
led to the identification of a sequence upstream (-166 to-149 bp) of TATA box, which was light responsive and was, therefore, conveniently called light responsive element (LRE).
This LRE sequence could also regulate genes other than rbcS
gene, so that the activity of these unrelated foreign genes became light, organ and age specific in a manner similar to rbcS
gene (high expression in leaves, low expression in stem and no expression in roots). LRE was shown to have three regions, box
II and box III
and it was shown that box II region also had a negative regulatory element. This LRE also seems to be repetitive (with two repeats) and each repeat unit can mediate light responsiveness and organ specificity. However, it has also been shown that both repeats of LRE are required for full expression of the rbcS
gene in youngest leaves of tobacco plants.
It has also been shown using transgenic plants, that a different sequence upstream to rbcS
is responsible for age responsiveness.
For example, it was shown that the region from -410 to -166 bp is essential for high level of expression in imuiature leaves, but is not important for light responsive expression in mature leaves, where ;he region -166 to -149 bp was most important.
Gene for chlorophyll a/b binding protein (Cab).
Examples of the transfer of Cab
gene from pea, wheat and Nicotiana plumbaginifolia
are listed in Table 37.4. In this case it was shown that a 247 bp sequence for pea Cab
AB80 (-347 to -100) and a 268 bp sequence (-357 to -89) for wheat Cab-l,
when separately fused with heterologous TATA sequence fused to neomycin phosphotransferase (NPT II)
coding sequences, conferred on this coding sequence a light responsive expression. This regulatory sequence when present induced expression of NPTII by red light and the expression could be reversed by a flash of far-red light as observed in phytochrome mediated expression. In another experiment, negative elements suppressing expression were also identified, when Cab-
Egene of Nicotiana plumbaginifolia
was studied in a similar manner in transgenic plants.
Heat shock genes.
Thermal stress induces the transcription of a small set of heat shock genes in diverse organisms, and also reduces the expression of many other vital genes. The upstream region of heat shock genes regulating their expression is highly conserved, so that when a gene construct carrying nptII reporter gene,
fused with upstream region of heat shock gene hsp
was introduced into tobacco, induction of NPTII expression due to heat shock was comparable to that of endogenous plant heat shock genes (inducible in all organs except pollen). Similarly, in maize a homologue of above gene, hsp70
with 1.1 kilobases of upstream sequence, when introduced in transgenic petunia, exhibited heat inducibility.
Alcohol dehydrogenase gene (ADH).
Alcohol dehydrogenase gene expresses under anaerobic conditions leading to oxygen deprivation. Using transgenic plants, it could be shown that regulatory sequences upstream to ADH
gene from maize lead to suppression (downregulation) of gene activity under aerobic conditions, this activity being normal and uninducible under anaerobic conditions. The inability of the ADH
upstream regulatory sequences to induce gene expression became evident when these sequences failed to induce chloramphenicol acetytransferase (CAT)
expression under aerobic as well as anaerobic conditions in transgenic tobacco plants. Other enhancer sequences (from octopine synthase
gene), when transferred at a position upstream of ADH
gene, led to induction under anaerobic conditions. Therefore, under normal conditions, ADH
activity is inhibited under aerobic conditions due to negative control and expression is promoted under anaerobic conditions simply due to the absence of a negative control rather than the presence of positive induction.
Genes for seed storage proteins in legumes and cereals.
The genes for seed storage proteins provide an excellent example of a cell and tissue specific expression. Using transgenic plants, sequences could be identified which were responsible for tissue or cell specific expression of these genes. For instance, when wheat glutenin
gene or barley hordein
gene (with its regulatory sequence) was fused to chloramphenicol acetyltransferase (CAT)
gene and introduced into tobacco, CAT activity was found only in the endosperm. Another reporter gene utilized for the same purpose is β-glucuronidase (GUS)
gene, which also showed endosperm specific expression when fused with a wheat glutenin gene (with its regulatory sequence). The regulatory sequence thus identified was called endosperm box
of the storage protein genes.
EPSP Synthase gene.
5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS) is an enzyme involved in aromatic amino acid biosynthetic pathway and is the target of glyphosate based herbicides.
In petunia, enzyme is synthesized in flowers at a high level and is hardly found in leaves, although in tomato it is synthesized both in flowers and leaves. Transgenic tobacco plants carrying CAT
gene or GUS gene fused with deletion mutant forms of EPSPS gene were analysed and it was concluded that sequences between -1800 to -800 of petunia EPSPS gene were responsible for tissue specific expression of this gene.