Author Archives: Pia Smets

06/8/14

Simulating climates in growth chambers – The AdapTree project

This post is part of the series Simulating Climates in Growth Chambers.

The AdapTree project evaluates the genetic and phenoptyic variation of two commercially important conifers in western Canada, interior spruce (Picea glauca, P. engelmannii, and their natural hybrids) and lodgepole pine (Pinus contorta). More than 580 seed sources were grown under controlled climate conditions to quantify genetic diversity and geographic structure for adaptive traits such as phenology, frost hardiness, seedling growth, and response to drought and heat. Concurrently, sequence capture and resequencing of much of the exome for ~600 individuals of each species reveals genetic variation, some of which is associated with this adaptation. Around 5,000 additional individuals per species growing in various other controlled climate regimes and outdoor common gardens will then be genotyped using a cheaper SNP array. All of these markers will be tested for a potential role in local adaptation to climate through 1) association with climate-relevant phenotypes; and 2) gene-environment correlations. The SNPs with evidence of local adaptation will then be used to evaluate the suitability of populations to future climates. Field-based validation studies have been established to confirm the genomic results.

Location of the target simulated climates on a map.

Location of the target simulated climates on a map.

To bring out the differences in adaptive characteristics of populations from all over British Columbia and Alberta, three temperature regimes were developed to represent four different climates with mean annual temperatures (MAT) of 1, 6 and 11 °C (all well watered), as well as an MAT 11 dry climate.
Realistic climates were needed to yield realistic bud break and bud set data in growth chambers. The photoperiod regime was identical for all plants and day length corresponds to that at 54.5°N at the relevant time of the season. Time constraints on the project necessitated germination modification and growing season compaction to reach the desired plant sizes quickly for all experiments. Not all experiments could be grown under fully controlled conditions, and some of the plants grown in the greenhouse were out of sync with nature yet needed to be planted outdoors in the following season. Their blackout regime gave us trouble, since covering them up to keep light out caused conditions ideal for fungal growth. During the second growing season, simulated drought was applied in cycles to the MAT11 dry treatment. Half of the plants in MAT11 wet and half of the plants in MAT11 dry were also subjected to a heat wave in the middle of summer. Plants were well-watered and chlorophyll fluorescence was measured to evaluate plant stress as a consequence of the heat wave. Carbon isotope composition was used to evaluate plant response to drought integrated over the growing season. At the end of the second growing season, cold hardiness measurements required us to simulate real winter, and not just a chilling period. The night frost pre-treatments were successful and good cold hardiness data were obtained. After this, the plants could be destructively sampled for dry weights. This required washing the soil off the roots. Thanks to our foresight in using plant cones during the first season (see root washing) we were able to separate the roots even for the largest (MAT 6 and 11) plants and after two seasons of growth.

Further information about the AdapTree project can be found on the website.

 

06/7/14

Simulating climates in growth chambers- Germination modification

This post is part of the series Simulating Climates in Growth Chambers.

In some cases, quick and relatively uniform germination is desirable even in the coldest treatment, to reduce noise among populations. In our case, a minimum plant size was also needed for the plants in the coldest treatment to allow for the harvest of fresh green material, at the beginning of the second season, for DNA extraction without impacting growth or phenology. This required starting out with a uniform ‘greenhouse’ regime, which gradually diverged and morphed into climates of 2, 6 and 10 °C MAT. The second season started with the ‘real’ target climates of MAT 1, 6 and 11°C.

Modification of germination temperatures to speed up germination, achieve necessary plant sizes and reduce noise. On the left: the first 14 weeks gradually evolve from a ‘gree

Modification of germination temperatures to speed up germination, achieve necessary plant sizes and reduce noise. On the left: the first 14 weeks gradually evolve from a ‘greenhouse’ regime to a real MAT 6°C regime. Right: the last ten weeks are unmodified.

Gradual diversification of temperature regimes in the first season

Gradual diversification of temperature regimes in the first season

06/6/14

Simulating climates in growth chambers- Growing season compaction and stimulating bud set without a growth chamber

This post is part of the series Simulating Climates in Growth Chambers.

Much more difficult is preparing the plants for winter when limited control over day light and temperature is available. If your plants have been growing in the greenhouse under warm and constant temperatures, and are still growing happily, how can you make them set bud and prepare them for a second season in the growth chamber? Commercial growers use a procedure called black-out in mid or late summer. Day length is reduced to about 10 hours (in British Columbia) by shading the plants using black cloth, e.g. the cover goes on at 7:00 pm and is removed the following morning at 9:00 am. Usually this is accompanied by a lower fertilizer concentration (esp. Nitrogen), a reduction in moisture levels and -if possible- a slight drop in temperatures to cause mild (but not severe) stress. Blackout is not as easy to apply as it sounds, since good ventilation is desired. The treatment is applied for 7 to 10 days, and resulting bud formation should begin to be visible in 2-3 weeks. Though it is not a very natural procedure, firm plants and firm buds can be obtained this way.

Simply moving plants from 22°C (greenhouse) into cool temperatures (2°C) will not have the same effect: while the signal is clear, the biochemical reactions of the plant are slowed down at low temperatures. The only way to avoid this is to find a place where you have more control over temperature and day length, or are in sync with the seasons outdoors.

Growing season compaction

We reduced the length of the first growing season from 25 to 17 weeks by reducing phase A from 3 to 2 days, and phase B from 4 to 3 days. This resulted in only a slight change in average temperature. This not only saved some time, it also reduced the maximum size of the plants in the warmest treatments. As a result, competition in the second season was reduced below levels that would lead to widespread mortality. At the same time, it still took the plants through all the physiological stages and prepared them for the climates they would experience in the second season.

06/5/14

Simulating climates in growth chambers – Simulating heat waves

This post is part of the series Simulating Climates in Growth Chambers.

We are especially interested in plant response to stressful events.

We wanted to measure response to extreme heat using fluorescence, so we needed to superimpose a heat wave. The question became then: what does a realistic heat wave look like? The definition of a heat wave is “a prolonged period of excessively hot weather”. Definitions vary with country and region, and depend on the baseline climate of the area. Heat waves are often defined in terms of the impact they have on human health, not on plants. The World Meteorological Organization defines a heat wave as a sequence of >5 days where the daily maximum exceeds the average maximum by 5 °C (Wikipedia). De Boeck et al. (2010)1 analysed

characteristics of Western European heat waves. They defined a heat wave as a period of 7 days where temperatures reached higher than 90% of Tmax, where Tmax is the daily maximum determined from weather data and is a different number for each day of the year. The reference period is 1961-1990, so daily weather station data for 1961-1990 were needed for their method. We had neither the time nor the data for such an approach, but found several references to a duration of between 5 and 7 days for a heat wave.

Because of the background climate in our experiments, I resorted to the following solution. At the warmest time of the season (week 15), the warm (A) and cool (B) phases were swapped, resulting in a warm period of six days (15A+16A). Temperatures were increased by 5°C to make this six-day period “hot” instead of just warm. All plants were well-watered before the start of the heat wave (Fig. 6, pine dry, week 16: even the dry treatment received a full dose of water). Although in nature heat waves have drought stress associated with them, we were afraid to create conditions which were lethal rather than stressful. We simply couldn’t afford to lose half of the plants.

Temperature regimes during a six day heat wave were planned to otherwise minimally disrupt the background climate (MAT 11 °C).

Temperature regimes during a six day heat wave were planned to otherwise minimally disrupt the background climate (MAT 11 °C).

Plants were carefully observed for signs of stress. Lodgepole pine plants had already set bud and survived without any noticeable damage, and chlorophyll fluorescence was measured as a measure of stress on day 6 of the heat wave. Interior spruce plants were still actively growing and suffered visibly from this heat wave, with needles browning and tips dying. As a result, chlorophyll fluorescence measurements were completed on day 4 of the heat wave, after which the heat was reduced by 5°C to reduce plant death and data loss. In fact, spruce plants not subjected to either heat waves or drought also suffered from high temperatures in week 15-16, as evidenced by browning of needles.

 

1 De Boeck H., F.E. Dreesen, I.A. Janssens and I. Van Nijs. 2010. Climatic characteristics of heat waves and their simulation in plant experiments. Global Change Biology 16: 1992-2000.

06/4/14

Simulating climates in growth chambers – Simulating winter

This post is part of the series Simulating Climates in Growth Chambers.

It remains difficult to simulate realistic winters. The more expensive growth chambers will allow programmed temperatures of -2 °C at night (with lights off). Fluorescent and incandescent lights themselves generate heat and the cooling requirement is energy-demanding. LED lights are costly. While a temperature of -2°C is technically possible, I have found out the hard way that this may not actually be what you want. When submitting plants to cold temperatures during the day (+2°C) and freezing temperatures at night (-2°C) it has happened that my soil volume froze, and did not thaw during the day. This resulted in wilting. In nature, plants would form roots deeper down, which have access to soil water. In the chamber, soil volume is very shallow, and assumes the temperature of the surrounding air, while buffering only the highest and lowest extremes.

Our interest has mostly been in growth, so a six-week “chilling period” is applied after one growth season to encourage more uniform flushing in the second growing season. During the chilling period, temperatures are held at a constant 4°C and day length is reduced to 8 hours or less, under reduced light intensity. This has been sufficient in our case, since the plants had received the correct signals to set bud and prepare for winter by the time our simulated end of the growing season (“October 15”) arrived.

In certain situations it is necessary to have real frost. We found this was true for testing cold hardiness of pine and spruce. We use the electrolytic leakage method described in Hannerz et al. (1999)1. Day length is the most important trigger in signalling the end of the growing season and initiating dormancy. However, in nature these shorter days are correlated with cooling temperatures. When the factors are uncoupled in the growth chamber, we find that the cooler temperatures are a required component of the acclimation procedure. We have successfully induced hardiness by subjecting plants which had already set bud to brief periods of night frost. To this aim, we gradually, over a period of 4 days, dropped night temperatures to a minimum of -2°C, until we kept it there for two hours.

Temperature regimes for a pre-treatment of light night frost to induce cold tolerance. Grey are baseline temperatures for resp. part A and B of the week, NF 1 to 4 are the four nights of frost. NF 0 (red, preceding NF1) was introduced for MAT6 and MAT11 because of their high nighttime minima.

Temperature regimes for a pre-treatment of light night frost to induce cold tolerance. Grey are baseline temperatures for resp. part A and B of the week, NF 1 to 4 are the four nights of frost. NF 0 (red, preceding NF1) was introduced for MAT6 and MAT11 because of their high nighttime minima.

A week after this acclimation took place, pretests revealed that the plants had responded sufficiently to reveal the population signal we were interested in, and yield suitable testing temperatures. Full tests were scheduled for the second week after the frost. Higher daytime temperatures (maxima between 23 and 30 °C) did not undo or mask the nighttime frost signal, and the soil never risked freezing with these brief exposures.

Effect of pre-treatment of light night frost to induce cold tolerance on lodgepole pine.

Effect of pre-treatment of light night frost to induce cold tolerance on lodgepole pine.

1 Hannerz, M., S.N. Aitken, J.N. King, and S. Budge. 1999. Effects of genetic selection for growth on frost hardiness in western hemlock. Can. J. For. Res. 29: 509–516