Chapter 6: General Discussion & Conclusions

The heat shock response, although universal in purpose, has evolved differently among organisms. Between the plant and animal kingdoms, the most notable differences are apparent the changes in the structure and function of the regulatory transcription factors, the HSF family (discussed in Chapter 1). This translates into the differences in the magnitude and complement of expressed HSPs in response to different abiotic stresses. Such evolutionary modifications of HSPs are observed in organisms in all geographic locations, among all kingdoms and even in micro-environments such as parasite host relationships (reviewed by Feder and Hofmann, 1999). Fundamentally, these differences are distilled as specific distributions of HSPs within the cell, presence or absence of HSP family members during stress and control conditions, degrees of induction, HSP kinetics and life span, and HSP tissue specificity. Such universal dependence on HSPs illustrates the vital necessity of these proteins, but the nature of their activity is greatly dependent on the physiology and environment in which an organism lives.

The differentiation in the heat shock response is more complex for plants, perhaps due to their sessile nature as well as their evolutionary head start on animals. Thus the question of why plants have so many functional HSFs is still unanswered. Nover et al., (2001) has suggested an explanation for the formation of the different plant HSFs, through alternative splicing followed by specialization. Furthermore, the differentiation of HSF roles may be connected to various HSP activities during all types of stress and numerous cellular activities. Another possibility is that specific HSF family members have developed optimal activities or binding affinities in response to different stresses as was observed by Bharti et al., (2000). It is also possible that some HSFs are evolutionary remnants or are involved in dosage effects when forming HSF heterotrimers (Bharti et al., 2000). Since plants face many environmental challenges, a greater understanding of HSFs and the heat shock response may identify means whereby HSFs can be manipulated to enhance stress tolerance.

6.1 The Heat Shock Response in Alfalfa under Heat Stress
Based on the results of the present investigation, alfalfa responds characteristically to heat stress. After exposure to 41°C, alfalfa plants increased their transcript levels of HSFA4, HSP 86, HSP 18 and accumulated high levels of class 1 low molecular weight heat shock proteins (lmwHSPs). The striking similarity among the surveyed genes, most notable with HSF expression, was that there was a delay in expression in onset and a decrease in magnitude of transcripts in alfalfa cultivars from dormant to non-dormant (Figure 3.4). In the case of HSF, the more non-dormant the cultivar, the more delayed the response. Conversely, the more dormant the cultivar the more rapid the response in HSFA5 transcription. This indicated that there is a correlative relationship between the genetics of dormancy, to MsHSFA4’s activity and the heat shock response.

Alfalfa cultivars are specifically adapted to environments for which the plants were bred or evolved. Non-dormant varieties are more adapted to warmer and/or drier climates than dormant varieties. Thus in alfalfa, non-dormant cultivars may be less dependent on the heat shock response for heat protection, a biologically expensive mechanism. It may be advantageous for the plant to delay initiation of the heat shock response for as long as possible as other mechanisms for coping with heat stress are functioning. To date, there have been no studies regarding this HSF dormancy relationship to support this result. Nguyen et al., (1994) observed increased levels of lmwHSPs in cultivars of wheat that were heat tolerant as compared to cultivars that were less heat tolerant. In contrast a reverse relationship between heat tolerance and levels of HSP 18 transcripts or the expression of class 1 lmwHSPs in alfalfa was observed. Under heat stress, it is possible that this MsHSFA4 dormancy relationship is unique to alfalfa (see section 6.5).

Comparing the results from Chapter 3 and Chapter 4 revealed a correlative relationship between HSF gene expression and lmwHSP expression (Figure 6.1 and 6.2). All cultivars exhibited a positive correlation under heat and cold stress, when MsHSFA4 transcript levels increase so do the levels of lmwHSPs. The slope of this correlation is greater under cold stress than it is under heat stress (Figure 6.1 and 6.2). This indicates that MsHASA4 is a transcription factor that may be related to low temperature stress or plants that have evolved to cope with low temperature or short season environments. Partial support for this theory comes from Arabidopsis micro-array experiments. In surveying 8 200 expressed genes in Arabidopsis under a variety of non-heat stresses including cold stress, Cheong et al., (2002) found increases in transcripts of several HSP genes (including HSP 83, HSP 70, an HSP 40 member and several low molecular weight HSP transcripts) and two HSF genes (of the possible 21). Although only a proportion of expressed genes were detected, the presence of HSFs under cold and other stresses indicated that low temperature HSF induction observed in these experiments was not isolated to alfalfa. There may well be more HSFs involved and thus a specific HSF study is warranted.

6.2 The Heat Shock Response in Alfalfa under Cold Stress
Under cold stress, HSFA4, HSP18, HSP86 mRNAs and lmwHSP protein accumulations were all observed to be expressed to varying degrees. Transcript levels of MsHSFA4 were up regulated under low temperature stress. This work represents the first report of a cold induced HSF. Expression patterns between dormant and non-dormant cultivars exhibited a similar dormancy cultivar trend that was observed under heat stress. MsHSFA4 expression in the non-dormant cultivars was delayed in onset and magnitude as compared to the dormant cultivars.
Increased expression of HSP18, HSP83 and lmwHSPs were also detected under cold stress, but at much lower levels as compared to heat stress. There were no detectable differences among cultivars with the exception of the lmwHSPs which exhibited a variety of expression patterns. Among the dormant cultivars, the expression levels under cold were comparable to the levels observed under heat stress. In contrast, the non-dormant cultivars exhibited greater levels of expression under cold stress as compared to heat stress. This suggests that cultivars that are less adapted to cold may require a greater abundance of lmwHSPs for protection. This finding is not surprising, as several researchers have observed increased levels of HSPs in response to low temperature (Guy and Haskell, 1987; Neven et al., 1992; Anderson et al., 1993).

Comparison of HSF and lmwHSP expression under cold stress revealed a positive correlation as was observed under heat stress (Figure 6.2). However the slope of the linear relationship was greater under cold stress. This observation supports the theory that MsHSFA4 is the HSF involved in the activation of the heat shock response under low temperature stress and to a lesser degree, an activator under heat stress.

6.3 The Heat Shock Response in Alfalfa during the Fall
During the fall, all cultivars exhibited fluctuating expression patterns across all surveyed genes. The patterns generally paralleled each other through September and October up until a critical point around the 30th of October where expression patterns began to diverge. This critical point occurred on the first days of the fall that the average field ambient temperature was 0°C. Ukaji et al., (1999) observed a similar effect in accumulations of lmwHSP homologues in mulberry, in which lmwHSP protein levels dramatically increased by the beginning of November. In the present study, among the surveyed genes and proteins, non-dormant alfalfa cultivars showed significant increases as compared to the dormant cultivars after the 30, October. This trend was observed for all genes except for the lmwHSPs which exhibited high levels in the dormant cultivars as compared to the non-dormant cultivars. These results must be taken in the context that there are numerous stresses and cues in the field and that it is impossible to implicate temperature as the sole affecting factor in these experiments. However, since these genes are established as temperature sensitive, correlative data supports temperature as the strongest influencing stress.

The data suggest that the non-dormant cultivars were sensing the field low temperature stress and responded by increasing HSP gene transcription via HSFs but the production of HSP protein was inhibited, possibly by temperature, resulting in lower levels of HSP production. These experiments were a unique attempt to observe components of the heat shock response under field low temperature conditions and thus there is little support form the literature to validate these findings. However, due to mounting evidence implicating the activities of HSPs under controlled cold stress and the universal induction of HSPs under all types of stress, it is plausible that HSPs are also involved in field low temperature protection.

6.4 The Diurnal Effect of Alfalfa HSF
Analysis of MsHSFA4 revealed significant variations in expression levels in alfalfa throughout a 16/8 hr day/night and was identical across all cultivars. This is the first report of a diurnally regulated HSF. This result was not unexpected since HSPs themselves exhibit diurnal expression (Merquiol et al., 2002). In addition to temperature stress, HSPs are involved in numerous cellular processes (Parsell and Lindquist, 1993). They aid in the formation and maintenance of large protein complexes, specifically the rubisco complex (Li and Guy, 2001; Ivey III et al., 2000) and the photosystem complex (Georgieva, 1999). These protein complexes diurnally fluctuate, are constantly recycled and require for proper chaperoning. The possibility that this diurnal effect is indicative of global RNA fluctuation in alfalfa was ruled out by using a probe to another transcript (data not shown) which exhibited constant and unchanging transcript levels. Thus it is plausible that MsHSFA4 is involved in modulating the diurnal activities of some HSPs.

6.5 Differences in Expression of HS Genes among Alfalfa Cultivars
The chamber and field experiment exemplified the importance of genotype sampling and replication when assessing gene expression. The diurnal effect that was observed in all genes surveyed, exhibited no cultivar differences, but as soon as a stress was applied, dramatic cultivar differences appeared. The specific cultivar differences and dormancy relationships described above could only be determined by sampling germplasm spanning from dormant to non-dormant. This relationship has the potential for application in alfalfa cultivar production. Currently, measures of fall dormancy are obtained by observing the values for fall regrowth of different cultivars. This MsHSFA4 dormancy connection may have use as a molecular marker for assessing the fall dormancy of new alfalfa cultivars. This could be applied quickly in the laboratory instead of waiting an entire growing season to evaluate fall regrowth, a process that is susceptible to environmental conditions.

6.6 The Connection between Heat and Cold Stress
As was detailed in Chapter 1 (1.8 and 1.9), there are a number of studies reporting the increased expression of HSPs under a variety of low temperature stresses in different organisms. Whether this enhanced HSP level confers protection under low temperature stress is still up for debate. However. most researchers accept the hypothesis that the chaperone capabilities of HSPs are involved.

Under low temperature stress, only specific HSP genes are activated. This implies that the control mechanism for the heat shock response, the HSF family, has the ability to act in a gene specific manner under low temperature stress. Alternatively, these specific low temperature induced heat shock genes could be activated by another cold responsive regulatory system aside from the HSF family.

To answer this question, two key components of the cold induced heat shock response must be studied. First, a low temperature responsive transcription factor or other gene regulator that directly or indirectly activates specific HSP genes during low temperature stress must be identified. Second, if HSFs are the activators, the combination of HSF family members that are involved and/or how the HSF promoter interaction differs from heat stress must be confirmed.
The experiments on MsHSFA4 described in Chapter 3 dealt with the first component, identifying a cold responsive HSF. MsHSFA4 transcripts are induced under cold stress and MsHSFA4 is a functional activator of HSP gene transcription under non-stress and cold stress conditions (Chapter 3, 3.2.1). These results imply that HSFs, specifically but not solely class A4, are regulatory transcription factors that activate HSPs under cold stress in alfalfa. How the HSF can activate selective HSP genes is still a mystery. This selectivity may be a physical characteristic of the HSFs, a dosage effect of hetero- versus homotrimers, regulation of the HSF transcript or protein or an inherent characteristic of the promoters of the selective heat shock genes.

Research to answer these questions will help develop a unifying theory of how the heat shock response, or better termed the stress response, functions under all temperature stresses. It may also shed light on how HSPs work in consort with other stress response mechanisms to protect against damaging effects of both high and low temperature stress.

6.7 The Utility of Engineering HSF to Protect Against Temperature Stress
These results are not yet available to the public due to issues involving intellectual property.
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