Environmentally Induced Heritable Changes in Flax

1. Growth of flax under inducing conditions.

1. 5″ pots are filled with soil and firmed.
2. Three seeds per pot are planted ¼ inch deep and the soil firmed over the seeds.
3. The pots are then watered with 100ml of the appropriate nutrient solutions
   1. The non-inducing control (C) conditions use a 1/10^th strength Miracle-Gro every week. The high nutrient condition (NPK treatment) had full strength Miracle Grow applied weekly. Low nutrient – plants only had tap water applied throughout their growth. (Durrant 1971, Cullis 1980).
4. The plants are grown under natural light during the summer. During the winter they are grown under sodium lights with a 16/8 hour light/dark cycle.
5. At weekly intervals leaves and meristems are collected.
6. The plant material is quick frozen in liquid nitrogen.

Results

The three genotypes grow at different relative rates depending on the conditions (Figure 1). Therefore under control conditions the Pl line grows best with L being more vigorous than S (Figure 1a). Under low nutrients (water), S grows better than either L or Pl (Figure 1b). Under high nutrient (NPK) L grows better than Pl which only grows slightly better than S (Figure 1c). A comparison between each of the lines grown under the three differing treatments is shown in Figure 1 d – f. Pl grows best under control conditions (Figure 1d), L best under high nutrient (Figure 1e) and S similarly under both high nutrient and control conditions (Figure 1f).

These data show that the three lines can be differentiated based on their growth under the three environments. Of note is that Pl grows best under the control conditions while L grows best under the NPK conditions (under which it was induced). S grows about the same in all three conditions, that is, it is not able to take advantage of improved nutrient conditions but is better able to grow under very low nutrient conditions.

The growth parameters that are relevant to the differentiation of the plants include the plant height (and associated with this the internode length, that is the distance between each leaf), the leaf size and the degree of branching. For Pl under control conditions the plant is tall with branches and the leaves are large and dark green. Under low nutrients the plant is very short, the distance between each leaf is also very short and the leaves are small and light green. The leaves at the tip are darker green than those lower down the stem. Under high nutrients the plant looks very similar to that under control conditions except that both the main stem and the side shoots are shorter than in the control plants. For the large genotroph the plants look very similar to those of Pl except that the most vigorous growth in under high nutrients. Again under low nutrients the plant is very short and has light green leaves. The S genotroph grows similarly in all three environments although the leaves are lighter green under the low nutrients. However, in the low nutrient conditions the S genotroph is much more vigorous than either of the Pl or L lines.

2. RNA and DNA extractions from flax meristems and leaves are done separately. The Roche total RNA prep kit was used for the RNA extractions and the Qiagen DNeasy Plant DNA prep kit was used for the DNA extractions.

RNA Extraction

1. 3 meristems plus some surrounding leaves are moved from storage cryovial into eppendorf tube and placed into liquid nitrogen until ready to be ground.
2. Sterile sand is added to tube and tissue is ground using a micropestle until fine.
3. 400 ul lysis/binding buffer from Roche total RNA prep kit is added and tissue is further ground until no clumps are visible.
4. Sample is vortexed for 15 sec to aid lysis and then spun for 1 min at 13,000 rpm to collect sand and any debris.
5. Supernatant is added to spin column and the rest of the RNA prep kit instructions are followed as directed.

DNAse treatment

1. 1/10 RNA sample volume of 10X DNAse buffer is added.
2. 30 units of DNAse is added and reaction is incubated at 37°C for 30 min and 70°C for 5 min.

Reverse Transcription

1. Applied Biosystems RNA to cDNA kit is used as per directions, reaction incubated at 37°C for 1 hour and 95°C for 5 minutes.

The cDNA is then used in PCR or for qPCR

qPCR

qPCR was performed using an ABI Step One system. All assays were performed in triplicate on each run. The actin gene was used as a copy number control as the best available housekeeping gene.

1. The appropriate primer pair was added to each tube in 1μl.
2. The cDNA was diluted to the appropriate concentration and 9μl added to each tube
3. 10μl of the 2x Power SYBR Green PCR MasterMix (ABI) and was added to each tube
4. The program amplification program was:
   • 10 minutes at 95°C
   • 40 cycles of 15sec at 95°C, 1 min at 60°C
   
   Thermal denaturation step to verify amplification specificity
5. The relative expression levels were determined by the value of 2(^{CTunknown – CTactin}).

DNA preparation

1. Buffer AP1 from Qiagen DNeasy Plant DNA prep kit is added to 6 leaves with sterile sand in a microfuge tube. The tissue is ground with micropestle until no clumps are visible.
2. The kit instructions are followed as directed.

Results

PCR amplification from the DNA isolated from leaves at various positions along the stem demonstrates that the appearance of LIS-1 occurs while the plants are growing under inducing conditions (4). The qPCR results show that the presence of LIS-1 (or other genomic changes associated with the induction) affects the expression of genes in the immediate vicinity of LIS-1 (Figure 2). The six genes shown in Figure 2 are all differentially expressed in Pl and S with four having higher expression in PL (without LIS-1) and two having higher expression in S (which does have LIS-1). Panel 1 and 2 are the two genes closest to the insertion point of LIS-1. The expression of these gene s in the large genotroph (which has had changes induced but does not have LIS-1) and the expression under different growth conditions will be needed to determine any causal relationship between LIS-1 and the expression changes.

Figure 1. Pl, L and S grown under three different nutrient regimes. Panels a – control, b – low nutrients and c – NPK. d – Pl, e – S, f – L. Panels d – f from left to right: low nutrients, control and NPK. D – Pl, e – L, F – S.

Figure 2. Gene expression around the point of insertion of LIS-1. Gene 1 (Inhibitor of growth) is immediately 5′ to LIs1 and the gene 2 (Kip-related cyclin-dependent kinase inhibitor id 3″ of LIS-1. The other genes are all along the same scaffold (~500kb) built from the complete flax genome sequence. Please click here to see a larger figure.

Figure 3. PCR amplifications of DNAs extracted from individual leaves of plants of Stormont Cirrus grown under low nutrient conditions. The PCR products were separated on a 2% agarose-TBE gel. The uninserted site is shown in Panel a, the two ends of the inserted site are shown in Panels b and c. Leaf samples 1 – 6 were isolated at weekly intervals with sample 1 being the earliest after sowing.