Chapter 5: Engineering an HSF in Alfalfa

5.1 Introduction
The creation of transgenic plants with altered HSF expression have been an important tool used to broaden our understanding of how plant HSFs function to regulate the heat shock response. Researchers have begun to turn their attention to the possible exploitation of HSFs in agriculture. Since HSPs are involved in helping cells cope with the effects of numerous stresses, the heat shock response is a good candidate for engineering stress tolerant plants.
A number of experiments have demonstrated that giving plants a heat shock pretreatment, thereby allowing HSPs to accumulate, induces protection against different types of cold stress (Ukaji et al., 1999; Cabané et al., 1993; Collins et al., 1993; Neven et al., 1992; Lurie and Klein, 1991). This helped formulate the hypothesis that if the heat shock response is activated in the absence of stress, then the plant would be primed and could better tolerate a subsequent related or unrelated stressful condition.

Logically, the next step is to assess the potential of using HSFs to alter the activity of the heat shock response under non-stress conditions. In 1995, Lee et al., reported the first transgenic Arabidopsis engineered with an overexpressed HSF A1 which activated the expression of HSPs under non-stress conditions. These engineered plants possessed increased basal tolerance to heat stress. Prändl et al. (1998) performed similar experiments in which an overexpressed HSF A3, in Arabidopsis, derepressed the heat shock response under non-stress conditions and increased tolerance to high temperature heat stress. These experiments demonstrate that is possible to overexpress HSF and engineer a constitutive heat shock response, and improve tolerance to stress. In wild type plants, HSPs must accumulate first to have their protective effect. For the engineered plants, the HSPs are already present, which reduced the lag time to accumulate enough HSPs to acquire their protective effect. This is an advantageous characteristic as many plants are exposed to rapid temperature changes that can often occur in field settings. Thus, engineering plants to pre-accumulate HSPs could have beneficial effects by providing protection against various field stresses.

A number of plants are vulnerable to rapid temperature reductions such as chilling and frost, as well as prolonged low temperature stress or over-wintering. Will overexpressing the heat shock response in plants induce protection against low temperature stress? Past research has revealed that HSPs are involved in protection to a variety of low temperature stresses (Chapter 1, 1.9). Thus having the HSPs pre-accumulate, or having the HSP system under the control of stress specific cold responsive promoters, could boost the chances of survival and/or minimize temperature-induced losses in yield. This type of HSF engineering might be most effective in plants that have been moved from regions of warmer climates to regions of colder climates, possibly extending the northern limits of agriculture or allowing the movement of tropical crops to temperate regions. Recently, Li et al., (2003) have demonstrated that under laboratory conditions the overexpression of AtHSFA1b in tomato enhanced tolerance to chilling stress. This experiment supports the hypothesis that an engineered overexpressed heat shock response can enhance low temperature tolerance.

It was hypothesized that constitutively increasing the basal level of HSPs through an engineered HSF would improve the ability of alfalfa plants to endure the effects of low temperature stress. This chapter details experiments designed to ascertain the effects of a constitutively expressed Arabidopsis class A4 HSF in alfalfa. The experiments were conducted to overexpress the heat shock response and observe the effects of HSP induction on winter survival, spring regrowth and plant biomass.

5.2 Materials and Methods

5.2.1 Binary Vector Construction

Transformation vectors were constructed using the binary vector pMW9805 built by Yang Wang (Bowley Lab, University of Guelph). The pMW9805 was constructed using the pART 27 vector (14Kb) as a starting backbone (Figure 5.1) and incorporating an npt II (neomycin phosphotransferase II) gene for resistance to kanamycin. The inserted HSF, AtHSFA4a (GenBank accession number U68561) was driven by the super-promoter (Ni et al., 1995) which was chosen as several studies have shown it to exhibit significantly higher levels of expression as compared to the traditional full length cauliflower mosaic virus derived promoter (CaMV 35S). The super-promoter consists of 3 tandem octopine synthase (ocs) enhancers (Figure 5.1, Aocs) upstream from the mannopine synthase (mas) enhancer (Figure 5.1, Amas) and mas promoter (Figure 5.1, Pmass).

The HSF cDNA, AtHSFA4a from Aribidopsis thaliana, provided by Dr. Eva Cznerecka-Verner (University of Florida) was cloned into the pBI121 vector (Clonetech). The cDNA was obtained from the pBI121-AtHSFA4a vector through PCR using Expand™ high fidelity polymerase (Roche). Due to sequencing errors engineered into the PCR primers, which prevented sticky end ligation, a TA cloning procedure was employed. The pMW9805 vector was cut open at an Xba I site and blunt ended with the Klenow fragment of DNA polymerase (Invitrogen). The vector was gel purified and then incubated at 37°C for 6 hrs in the presence of Taq DNA polymerase and 0.5 mM dTTP. This resulted in preferential addition of single thiamine bases to the 3’ ends of the vector. The AtHSFA4a PCR fragment was gel purified and required no further processing for TA ligation as the Taq DNA polymerase had already added 3’ A overhangs. The AtHSFA4a fragment was then combined with the T overhang processed pMW9805 vector in the presence of T4 DNA Ligase and incubated overnight at 16°C. The ligation reaction was then electroporated into competent DH5a E. coli and plated on LB spectinomycin plates. Plasmids were extracted from positive colonies and fragment insertion and fragment orientation were confirmed through Not I restriction digests. These results were reconfirmed through PCR and DNA sequencing of the T-DNA region of the isolated binary vectors.

The newly engineered vectors were named pAtHSF21-9901s for the vector containing the AtHSFA4a cDNA in the sense orientation and pAtHSF21-9902as for the vector containing the antisense orientation. DH5a -80°C bacterial stocks were prepared for each vector and stored at -80°C.

5.2.2 Agrobacterium Mediated Transformation
The pAtHSF21-9901 and pAtHSF21-9902as binary vectors were separately inserted via electroporation into the Agrobacterium tumefaciens C58C1 strain containing the pMp90 helper plasmid. Agrobacterium containing both the binary vector and the helper plasmid were selected for on LB plates containing spectinomycin (selecting for pAtHSF21 vectors) and gentimycin (selecting for pMp90 helper plasmids). A 50 ml liquid culture of a positive colony was grown to an OD at 600 nm of 1, the level required for transformation.
For transformation the alfalfa N4-4-2 genotype (developed by the Bowley Lab, University of Guelph) was employed as it produces large numbers of somatic embryos in tissue culture. Young petiole tissue was harvested from greenhouse grown plants and 1 cm sections were surface sterilized by submerging in 75% (v/v) ethanol for 30 sec, followed by transferring to 4% (w/v) calcium hypochlorite for 20 min and finally rinsing three times in sterile ddH2O. Transformation
Prepared alfalfa petioles (approximately 200 per construct) were incubated in the Agrobacterium culture for 1 min in the dark at 28°C. The petioles were then transferred to induction medium (Appendix B, SHK growth medium with the addition of 1 mg/l of 2-4-D) plates containing 100 uM acetosyringinone and incubated in the dark at 25°C for 3 days. Tissue Culture & Selection
After Agrobacterium inoculation, the petioles were rinsed in a salt solution (2.2 g/L MS salt, pH 5.8) to remove excess Agrobacterium. The petioles were then transferred to selection medium (Appendix B, SHK growth medium) (10 petioles per 80mm plate) containing 500 mg/L claforan and 50 mg/L kanamycin, sealed with parafilm and stored in a 28°C incubator with a 16-hour photoperiod. Cultures were transferred to fresh plates every 10 days.

Developing embryos were visible approximately 2 months after transformation and were transferred to 50 ml cylindrical glass culture tubes, containing regeneration medium (Appendix B, SHK growth medium), given a numerical designation and stored in a 28°C incubator with a 16-hour photoperiod. Once the plantlets reached 5 cm in height and a significant root system was established, they were transferred to turface (crushed clay) solid medium and placed in the greenhouse, under automated watering (2x daily for 5 min) supplemented nutrient fertilizer (Appendix F). For the first three days after transfer a transparent plastic cup was placed over the plantlets to prevent dehydration.

5.2.3 Confirmation of Transgene Insertion PCR
A single leaflet was excised from recovered plantlets for PCR analysis. A crude DNA extraction was performed using the FastPrep 120 instrument (BIO 101, Carlsbad, California) and the following the “mangonel” DNA extraction protocol (Bowley Lab, University of Guelph). A leaflet was added to a Fastprep tube on top of a Fastprep round ceramic bead and sandwiched beneath a Fastprep ceramic cylindrical bead. To each tube 500 ul of homogenizing buffer (Appendix B) was added, tightly capped and placed in the FastPrep machine and ground for 15 sec at 4 m/s. After grinding, the tubes were placed on ice to settle the foaming and then centrifuged at 8000xg for 6 min to pellet the cell debris. The supernatant, approximately 300 ul, was transferred to a new 1.5 ml microtube containing 600 ul 100% ethanol, inverted to mix and let stand at room temperature for 2 min. The tubes were then spun at maximum speed for three min to pellet the DNA. The supernatant was discarded and the pellet was inverted to drain and air dried for 5 min on a flow bench. The pellet was loosened with a pipette tip and resuspended in 100 ul of ddH2O (pH 8.0) and incubated at 4°C overnight.

PCR confirmation of transgene insertion was accomplished using the AtHSF-3a and AtHSF-3b primer pair (Appendix A) specific to the ORF of AtHSFA4a. Amplification occurred under the cycling conditions of denaturing at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C (using Taq DNA polymerase, Roche) for 1.5 min for 32 cycles. PCR products were analyzed on an 0.8% agarose gel stained with ethidium bromide. Southern Blot Hybridization
Isolation of genomic DNA for southern blot hybridizations was performed using the Trebuchet gDNA extraction protocol described in Chapter 2, 2.2.5. A DIG labeled DNA probe specific to the AtHSFA4a sequence engineered into the binary transformation vector (Chapter 5, 5.2.1) was prepared using the AtHSF-1a and AtHSF-1b primer pair (Appendix A) and the PCR synthesis reaction was completed as described in Chapter 2, 2.2.8. The Southern blot hybridization was performed as described in Chapter 2, 2.2.9 with the following change. Genomic DNA samples were digested with Eco RV which produced one cut inside the T-DNA insert and second cut outside the T-DNA region in the alfalfa genome. The critical fragment threshold for full length T-DNA insertion was 3000 bp. Northern Blot Hybridization
RNA extractions for northern blot hybridizations were performed as described in Chapter 3, 3.2.7 with tissue sampled from shoot apical meristems and mature leaves. The probe used in the northern blot hybridization was designed to bind to the ORF of AtHSFA4a transgene, prepared using the AtHSF-3a and AtHSF-3b primer pair (Appendix A) and the PCR synthesis reaction was completed as described in Chapter 2, 2.2.8. The northern blot hybridization was performed as described in Chapter 3, 3.2.11. RT-PCR
RNA extractions for RT-PCRs were performed as described in Chapter 3, 3.2.7 with tissue sampled from shoot apical meristems and mature leaves. RT-PCR reactions were performed as described in Chapter 2, 2.2.11 using primers AtHSF-3a and AtHSF-3b (Appendix A).

5.2.4 Testcross and Transgene Inheritance
Based on the quality of positive RT-PCR analysis , six primary transgenics, three from pAtHSF21-9901s and three from pAtHSF21-9902as were testcrossed to a non-dormant line of alfalfa, designated Z98 10-16 (dormancy class 9) (Bowley lab, University, of Guelph). All F1 progeny produced were germinated and screened by PCR (using the AtHSF-3a and AtHSF-3b primer pair and following the methods described in Chapter 5, for transgene segregation. Observed segregation ratios were tested using a X2 goodness of fit test for a 1:1 ratio expected for a single transgene insertion. Since the family sizes were small, Fishers Exact Test was completed for the goodness of fit and the Type I error rate was set at 0.05.

5.2.5 Field Experiment To Primary Transgenics
All positive primary transgenics as determined by PCR and southern screening were clonally propagated and planted in a randomized complete block design with 3 replications. Experimental units were single rows, 50 cm long spaced 20 cm apart, containing 6 clonal propagules of a plant genotype. There were 27 experimental units in a replicate comprised of 26 primary transgenics, 13 contained the sense construct and 13 contained the antisense construct, and 4 experimental units of the control genotype N4-4-2 (Appendix K). The trial included plants at two sites on the 7 June, 2001 at the Elora research station in Elora, Ontario (43°39’ latitude, 80°25’ longitude, 376 m elevation) and on 4 June, 2001 at the New Liskeard Research Station in New Liskeard, Ontario (47°30’ latitude, 79°40’ longitude, 194 m elevation) (Appendix L). All plants were evaluated for percent winter survival, spring regrowth and plant biomass. Spring regrowth herbage yield was harvested on 14 June, 2002 in Elora and 17 June, 2002 in New Liskeard. In the fall, plants were excavated and sectioned into herbage, crown (5 cm) and root (15 cm) on 11 October, 2002 in Elora and 1 October, 2002 in New Liskeard. All harvested tissues were evaluated for dry weight after incubating in 50°C drying oven for 3 days.

Statistical analysis was performed using SAS (
statistical analysis software, version 8.0, Cary California). A variance analysis, combined over locations, was performed using the “proc mixed” procedure of SAS. Variance was partitioned into location, block(location), construct, plant(construct), location x construct, and location x plot(construct). Block(location) was assumed a random effect, with all other effects fixed . Construct means were compared using a t-test. The type I error rate was set at 0.05. F1 Transgenic Testcross Progeny
Six F1 families, C198, C199, C201, C202, C205 and C206 were used in the field experiment. Based on PCR analysis (Appendix H), plants from a given family were separated into two groups, those with the transgene and those without the transgene (Table 5.2). The plants were clonally propagated (through stem leaf cuttings) and planted in a split plot arrangement with three replicates. Main plots were families, sub-plots were classes (with or without the transgene) within the family. Experimental units consisted of a row of 18 plants with the rows spaced 7 cm apart. Rows within a main plot were spaced 15 cm apart. (Appendix K). The experiment was established on 7 June, 2001 at the Elora Research Station in Elora, Ontario (Appendix L). All plants were evaluated for percent winter survival, spring regrowth, and plant biomass. Spring regrowth herbage yield was harvested and winter survival determined on 14 June, 2002 in Elora. In the fall, plants were excavated and sectioned into herbage, crown (5 cm) and root (15 cm) on 11 October, 2002 and dried at 50°C for 3 days for dry weight determination.

Statistical analysis was performed using SAS (
statistical analysis software, version 8.0, Cary California). All data were subjected to a split plot variance analysis using the “proc mixed” procedure of SAS (statistical analysis software). For this analysis the variance was partitioned into block, construct, family(construct), construct x block, block x family(construct), class, class x construct, class x family(construct). The blocks, construct x block and block x family(construct) were random effects and all other effects were considered fixed. Means were compared using a t-test. The type I error rate was set at 0.05.

5.3 Results

5.3.1 Transgene Integration and Evaluation DNA analysis
All plants that grew in the presence of selection medium containing kanamycin were screened by PCR and Southern analysis. After the first round of selection, 35 embryogenic calli survived and each was given a numerical designation ranging from 1 to 35. Of the 35 calli that produced growing embryos only 26 survived the full selection phase, 13 involving the sense construct and 13 involving the antisense construct transformations. Of these 26 independent putative transgenics, 23 plants tested PCR positive for the presence of the AtHSFA4 transgene and 3 plants (HSFS 15, HSFS 17 and HSFAS 21) tested PCR negative (Table 5.1).
Southern analysis revealed a variety of insertion events (Figure 5.2). The critical size threshold for a full length T-DNA fragment insertion was 3000 bp based on the Eco RV digestion. Of the 13 sense transgenic plants, 6 plants had a single insertion, 5 plants had two insertions, 1 plant had a partial insertion and 1 plant had no insertion. Of the 13 antisense transgenic plants, 7 plants had a single insertion, 5 plants had two insertions and 1 plant had a partial insertion. Specifically, plants coded 7 and 21 were found to have the partial insertion event (Figure 5.2 and Table 5.1). Plant 15 was negative for PCR and negative on the Southern and is considered an escape. Plant 17 was PCR negative but Southern positive with a complete single insertion indicating that the PCR screen produced a false negative. Plant 21 was PCR negative, Southern positive but was found to have a partial insertion which explains the negative PCR result. In total, transformations produced 4% escape, 7.7% with single partial insertions events, 50% with single insertion events and 38.5% with two insertions events (Table 5.1). Of the PCR and Southern positive plants 11 individuals contain pAtHSF21-9901s (6 with single insertions and 5 with double insertions) and 12 individuals contain pAtHSF21-9902as (7 with single insertions and 5 with double insertions). The overall transformation efficiency, percent of explants generating transgenic events was 6.25%. RNA analysis
No signal could be detected on a northern analysis of the primary transgenics, suggesting that the transgenes were not expressed or were in too low a copy number to be detected by northern analysis. DIG Northern analysis, under optimal conditions can only detect transcripts in concentrations greater than 0.1 pg. Thus, for a more sensitive expression analysis, RT-PCR which can detect expression as low as single transcripts, was employed, and resulted in the detection of transgenic transcripts in a number of plants (Figure 5.3 and Table 5.1). This indicated there was a low level of transcription of the AtHSFA4a transgene in most of the transgenic plants. Transgene Segregation
All F1 seed produced from the test cross were germinated and screened by PCR for the presence of the AtHSFA4a transgene (Appendix H). All families with exception of C201 (generated from plant 18) exhibited no heterogeneity as determined through a X2 analysis, thus families from each construct were pooled (Table 5.2). The pooled ratio of plants with and without the transgene did not differ from 1:1. Therefore, the transgene segregated as a single Mendelian gene.

5.3.2 Field Experiment To Primary Transgenics
Analysis of the winter survival at Elora revealed survival rates of 90-100% where as survival at New Liskeard was approximately 50%. This is indicative of the difference in severity of the winter between the two locations. Pooling of the winter survival data from the six parental plants containing the sense construct, antisense construct and both compared to the N442 non-transgenic control revealed significant differences at both locations (Table 5.3). In Elora, antisense plants exhibited less winter survival at 92% as compared to the control plants of 98% (Table 5.3). In this aspect, the sense plants were statistically no different than the control plants. In New Liskeard, sense plants had winter survival at 58.6%, the control plants had survival at 50.9% and the antisense plants had survival at 46%. These results for sense and antisense transgenics were statistically different from the control plants.

A variance analysis of yield data from Elora and New Liskeard indicated that there were no interactions with location. Herbage yield for spring regrowth revealed no significant differences. However, plant biomass (herbage, crown and roots) revealed significant differences between sense and antisense plants (Figure 5.4). In a frequency distribution analysis of number of plants versus biomass exhibited shifts to higher plant yields among sense plants when compared to the non-transgenic N4-4-2 control (Figure 5.4). Among antisense plants, the frequency distributions showed lower yields as compared to the non-transgenic N4-4-2 control (Figure 5.4).

Analysis of the six primary transgenics (To)used as parents to produce transgenic progeny (F1)exhibited significant differences. Sense plants exhibited 30%, 1% and 44% yield increases in herbage, crown and root respectively as compared to the N4-4-2 non-transgenic control (Table 5.3). Plants containing the containing the antisense construct exhibited opposite trend, 9%, 30% and 6% lower yield in herbage, crown and root, respectively, as compared to the N4-4-2 non-transgenic control. These differences were most dramatic in the storage tissues (crown and root), which exhibited mean yields of 7.1 g (se = 0.36) in the sense plants, 5.4 g (se = 0.62) in the control plants and 4.6 g (se = 0.37) in the antisense plants (Table 5.3). F1 Progeny Transgenics
Analysis of the winter survival in Elora revealed no significant differences among any of the families. However, winter survival was approximately 10% less as compared to the parental lines (Table 5.3).

Analysis of the yields of spring regrowth revealed no significant differences among the transgenic progeny. However significant differences were found when the yield was analyzed by tissue (Table 5.3). No differences were detectable in herbage and crown tissue but differences between the sense and antisense were detectable in the root tissue. Analysis of the storage tissue, crown and root together, revealed significant effects due to the presence of the transgene. Progeny containing the sense construct were 9% higher in storage tissue yield as compared to siblings without the transgene. Progeny containing the antisense construct were 13% lower in the storage tissue yield as compared to siblings without the transgene.

5.4 Discussion
Analysis of the primary transgenic plants revealed that the presence of the sense AtHSFA4a construct increased plant yield (~30% biomass) and marginally improved winter survival as compared to the non-transgenic control plants. Conversely, the presence of the antisense AtHSFA4a construct reduced yield (~14% biomass) and slightly reduced winter survival as compared to the non-transgenic control plants. Analysis of the progeny revealed that this trait, although less dramatic and localized to the storage tissue, was heritable. The fact that this trait was heritable and not family specific, confirmed the transferred T-DNA as the source of the changes observed in the primary transgenics and not the result of effects due to the insertion event or the effects of passing through tissue culture. As previous studies have shown the NPT II selectable marker to has no effects on alfalfa yield (Dr. Steve Bowley, personal communication). If NPT II was having an effect, yields trends would have been identical between plants containing the sense and antisense constructs. Thus, the changes observed in this experiment were the direct result of the expression of the AtHSFA4 transgene.
Experimental error was ruled out as the cause of the lower levels of transgene expression. Although a class A4 HSF has never been transformed into plants before, it is unlikely the cause as other HSFs have been successfully transformed into other plants. The reduced expression levels were most likely a result of the super promoter used to drive the transgene. This promoter, produced from the same bacterial stock, had been used by several labs on the University of Guelph campus, all of which have subsequently reported problems with gene expression. The original sequence of the promoter could not be determined or located (even from the original source) and thus it was impossible to confirm if there were any mutations present. As other plants have been successfully engineered with overexpressing HSFs, the problem of transgene expression is most likely a result of an altered promoter or enhancer that left this particular copy of the super promoter with reduced levels of activity.

AtHSFA4 in the sense orientation, significantly increased the mass of herbage, crown and root in the primary transgenics and reduced it in the antisense orientation. As root and crown size are directly related to surviving winters (Schwab et al., 1996), this HSF promoted increase in size should improve overall plant survival over the coming winter. These plants exhibited small, yet statistically significant, increases in winter survival in the primary transgenics only, as compared to control plants from the previous winter. However, this limited winter survival may have been a result of the low level of transgene expression, a manifestation of insufficient sample size or the possibility that the beneficial effects of this HSF was occurring over the growing season, protecting against high and/or low temperature stress. These are questions to which only a prolonged study could provide answers. The effects of this overexpressed AtHSFA4a could have occurred at any point in the life of these transgenic plants, but its stress protective effects best manifested itself in plant yield at the end of the growing season.
Although parallel trends were observed in the F1 progeny, the degrees to which yield was increased was smaller. This was most likely a result of the parents being crossed to a non-dormant alfalfa line, which manifested itself in the decreased pooled yields of the progeny plants as compared to the pooled yield of the primary plants (Table 5.3). The effects of the of the non-dormant background were also evident in the overall winter survival which was 10% lower in the progeny as compared to the parents.

There are several reports in the literature of engineered HSFs increasing stress tolerance in plants. Prändl at al., (1998) engineered an AtHSFA3 that resulted in increased basal tolerance to heat stress in Arabidopsis and Li et al., (2003) observed chilling tolerance by overexpressing a AtHSFA1 in tomato. However, my investigation represents the first report of an engineered AtHSFA4 or any HSF that improved or, when inhibited, reduced plant yield. Further study into the abilities of this HSF could translate into a new alfalfa cultivars with increased yields and stress tolerance.

5.5 Conclusion
Although constitutively expressed AtHSFA4a levels were lower than the levels expected of an engineered transgene, this experiment clearly demonstrates the benefits of engineering an HSF. Expression of AtHSFA4a in alfalfa improved field stress tolerance and resulted in a significant and heritable increase in plant yield, specifically in the storage tissues.