10.3.4 Gene expression modified by external factors

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Molecular genetic approaches are also providing novel insights into how activation of expression of specific genes and plant gene regulation mechanisms allow plants to respond to environ-mental cues. As examples, let us consider some genes which are switched on in response to both drought and cold — two environmental stresses which limit the productivity and geographical distribution of many agriculturally important species.


Figure 10.33 Drought or cold stress induce expression of genes such as RD29A in Arabidopsis. After perception of the environmental stress by the plant cell, a signal is transduced via activation of a DNA-binding protein called CBF1, which then binds to specific sites known as drought response elements (DREs) on the RD29A promoter. This leads to enhanced transcription of the gene and ultimately to accumulation of RD29A protein which probably helps to protect the cell against drought or cold stress. The RD29A promoter also contains elements which respond to the stress hormone abscisic acid (ABREs). The DRE and ABRE elements probably function together to enhance the rate of transcription.

(see Yamaguchi-Shinozaki and Shinozaki 1994 et al. 1997 for more detail).

Analysis of mRNA from Arabidopsis plants exposed to severe drought resulted in cloning of a number of cDNAs (complementary DNAs) representing genes upregulated by such stress. Several of these showed both abscisic acid (ABA)-dependent and ABA-independent expression, including a gene designated RD29A. The promoter of RD29A was isolated, fused to the GUS reporter gene and transferred into tobacco. Exposing these transgenic plants to drought resulted in a 16-fold increase in GUS activity over basal levels. Deletion analysis revealed a 157 bp region of the RD29A promoter located between nucleotides –268 and –111, which contained drought-inducible elements. A gain-of-function experiment, involving fusion of this 157 bp DNA fragment to a minimal promoter/GUS construct then transfer into transgenic plants, confirmed the presence of drought-induced positive regulat-ory sequences in this portion of the promoter. More detailed molecular analysis identified two identical short DNA elements of 9 bp in length, with the sequence 5' TACCGACAT 3', in this region of the promoter. Subsequent mobility shift analysis indicated that the two 9 bp elements are important for the binding of regulatory proteins to the promoter of the RD29A gene in response to drought stress and also cold stress, and that they stimulated transcription of the reporter gene in transgenic plants exposed to these stresses. These elements were designated desiccation response elements (DREs). Interestingly, DREs do not respond to variations in levels of ABA, indicating the presence of an ABA-independent pathway which regulates expression of the RD29A gene. It is likely that the DREs function in synergy with other parts of the promoter of this gene, such as ABA response elements (ABRE; see Table 9.2), to enable expression of the RD29A gene in response to drought via both ABA-dependent and ABA-independent pathways (Yamaguchi-Shinozaki and Shinozaki 1994). Recent work has shown that the core sequence of DREs (5'CCGAC 3') is found in single or multiple copies in promoters of several cold/drought-induced genes in plants and researchers have used the element to recover a cDNA from Arabidopsis encoding a DNA-binding protein designated CBF1 (C repeat/DRE Binding Factor 1). This protein binds to the DRE in vitro and probably influences transcription of RD29A and also other genes in vivo in response to drought and also cold stress (Stockinger et al. 1997). Interestingly and importantly, CBF1 mRNA levels do not increase in response to drought or cold stress, raising instead the possibility of post-transcriptional activation of the protein, perhaps by phosphor-ylation. Activated CBF1 can then upregulate expression of a whole suite of stress-related genes, including RD29A, which encode proteins that appear to protect cellular components from desiccation or cold-induced damage. The whole process is depicted in Figure 10.33. The link between cold and drought stress relates to cold-induced dehydration, so it is not surprising to find features in common at the cell and molecular level in cold- and drought-tolerant plants.


Figure 10.34 Resurrection plants such as the grass Sporobolus stapfianus, are species with remarkable adaptations to enable survival through extended and severe droughts. Although leaves of droughted plants become almost totally desiccated, recover of full leaf function takes a matter of hours following resupply of water. Underlying changes in patterns of gene expression enable this survival strategy, and have some analogies with processes during seed maturation. Here, mRNA abundance is plotted for two genes, one upregulated and one downregulated in response to dehydration. Dehydrins (green bars) are hydrophilic proteins which probably protect cellular components from damage under conditions of low water status. Chlorophyll a/b binding protein (Cab) is a core component of photosynthetic function. Expression of Cab (white bars) declines rapidly during dehydration, and indicates the shutting down of photosynthesis. Both these genes are also responsive to ABA supplied to fully hydrated plants, so this hormone probably plays a key role in mediating the stress response. However, there are other dehydration-induced genes which are not affected by ABA.


Figure 10.35 Environmental influences on products of gene expression can also be studied by monitoring protein levels. In this example, selective degradation of phytochrome A protein (phyA) occurs in response to exposure of dark-grown plant tissues to light. PhyA concentration is known to decline rapidly due to light instability, but the mechanism depends on conformational changes when the molecule is converted by light absorption from its stable Pr (red absorbing) to its unstable (far-red absorbing) form. This results in exposure of ubiquitin (Ub) binding sites. Ubiquitn is a small protein that 'tags' other proteins that are destined for proteolytic breakdown. A rapid accumulation of phyA-Ub complexes is detected using anti-Ub antibodies (●) and exponential decline in phyA concentration (○) is monitored with anti-phyA antibodies.

(Based on Clough and Vierstra 1997).

Dehydration is a normal feature of seed maturation and allows plant propagules to survive in suspended animation for extended periods. In a few species such as ‘resurrection’ plants, remarkably similar desiccation processes can occur in vegetative tissues and confer tolerance to severe and extended droughts (see Feature essay 15.1). When water is supplied again, the leaves rehydrate and resume normal functioning. The underlying changes in gene expression have strong parallels between seeds and resurrection grasses such as Sporobolus stapfianus. Control of these genes may ultimately lead to novel techniques for generating greater drought tolerance in crop and pasture plants. Here, we show changes in expression of two of the many genes involved during dehydration (Figure 10.34). One of these codes for a dehydrin gene whose mRNA accumulates as water content declines. Levels of mRNA are also responsive to ABA, the hormone that mediates many drought signals in plants. The other gene, encoding chlorophyll a/b binding protein, behaves in an opposite manner to the dehydrin and is downregulated both by decreasing water content and by ABA. This mirrors the loss of photosynthetic function during periods of drought. As with RD29A in Arabidopsis, some other drought-responsive genes are not regulated by ABA, which again suggests presence of at least two drought-signalling pathways.

Other environmental factors also induce changes in gene expression. For example, perception of light quantity and spectral composition involves phytochromes. One type, phytochrome A (phyA), is present in high concentrations in dark-grown plants but becomes extremely unstable after exposure to light converts it to the far-red (Pfr) form (see Section 8.4). The speed of degradation relates to presence on the phyA molecule of a site to which ubiquitin can bind. Ubiquitins are a class of proteins which ‘tag’ other proteins which are destined for degradation. The exponential decline in phyA, with a half-life of less than an hour, correlates strongly with abundance of phyA–ubiquitin complexes (Figure 10.35). Using techniques similar to deletion analysis of promoters, it has subsequently been shown that deletion of the ubiquitin attachment site converts the phyA molecule into a light-stable form (Clough and Vierstra 1997). This shows that external factors such as light can regulate the functioning of gene products at the protein (rather than the DNA/mRNA) level, and gives an insight into how cells can selectively degrade certain proteins.