Coping with Extreme Stress- Resurrection!

Susan JohnAdvisor: Dr. Karl H. Hasenstein

Department of BiologyUniversity of Louisiana at Lafayette

Effects of Water Stress on Plants

In response to scarcity of water plants wilt, and eventually die.

Resurrection ferns have a remarkable ability to tolerate dehydration by curling tightly and re-expand as soon as water (rain) becomes available.

Dehydrated fronds Rehydrated (resurrected)

Their ability to adapt to changing environment makes them an ideal model to study the effects of stress

Some plants however can tolerate water stressPolypodium polypodioides- Resurrection Fern

Drawback!There is no information on when different components

are synthesized during stress!

Various components listed below are known to be synthesized and suggested to be providing tolerance to desiccation tolerant plants

Osmoprotectants e.g. proline, glycerol Hormones e.g. Abscisic acid, salicylic acid Sugars e.g. sucrose

Early Studies on Stress Tolerance

A Challenging Question of our Research

What enables Polypodium to dry without dying?

Rehydrated frond Dehydrated frond

Polypodium fronds tolerate dehydration when dried slowly e.g. at 30 C, but they cannot sustain exposure to high temperatures e.g. 80 C

Hypothesis

To test our hypothesis we will expose Polypodium to different intensities of stress such as slow (30 C), intermediate (50 C) and fast (80 C) dehydration.

The effects of dehydration will be determined by measuring-1. Water status (Relative Water Content)2. Glycerol content (an osmoprotectant known to be

synthesized in response to stress) and3. Vitality (test whether dehydrated fronds are metabolically

active after rehydration)

Fresh fronds collected from oak trees

Fronds cut into half, floated on water for 12 h, weighed (rehydrated weight, WT)

½ rehydrated fronds placed in oven for drying at 30 C, 50 C,

and 80 C

Drying fronds removed from oven

at specific time intervals, and weighed (WD)

Method

½ rehydrated fronds dried at

80 C for 96 h to measure dry weight (WD)

2Glycerol content measured according to Sturgeon et al., 1979

3 Vitality test – Resazurin Assay

Relative Water Content (RWC)RWC% = Fresh weight -Dry Weight

Turgid weight – Dry weight × 100

1

Figure 1: (A) Relationship between percentage loss of relative water content and duration of dehydration. 30 C dried fronds took ~20 h to lose 90% of water compared to 2h taken by 50 C and 80 C dried fronds. Using log plot the time taken to lose 50% of water from 30 C, 50 C and 80 C dried fronds measured to be 5 h, 35 min and 27 min respectively. (B) At the end of 72 h of drying, the fronds were tightly curled, leathery to brittle to touch and appeared dark green to brown in color

Drying Profile of 30 C, 50 C and 80 C dried fronds

B

30 C

50 C

80 C

A Time (h)

RW

C %

Time (h)

ln (

RW

C%

)

Glycerol Content in 30 C, 50 C and 80 C dried fronds

Figure 2: Glycerol content of 30 C, 50 C and 80 C dried fronds decreased at the rate of 0.68, 0.59 and 1.57 mg h-1respectively during 12 h of dehydration. Between 24 - 72 h of dehydration, glycerol accumulated in 30 C dried fronds at a slow rate 0.13 mg h-1 and in 50 C and 80 C dried fronds the amount decreased at a rate of 0.18 and 0.09 mg h-1. There was no significant difference in glycerol content over the duration of dehydration (p >0.05). The data is represented as mean ± SE; N= 5 replicates per treatments

Time (h)

Gly

cero

l mg

g-1 D

W

Vitality Test of rehydrated 30 C, 50 C and 80 C dried fronds

Figure 3: Vitality of 30 C, 50 C and 80 C dried fronds were measured by incubating oven dried fronds with redox dye resazurin. The intensity of fluorescence corresponds to the metabolic state of rehydrating fronds. 30 C dried fronds showed higher intensity (metabolic activity) compared to 50 C and 80 C fronds. The intensities of 50 C and 80 C measured zero after 20 h of dehydration, suggesting no metabolic activity. Fluorescence was measured ( 530 nm excitation and 590 nm emission)N= 3 replicates, mean ± SE

Drying Duration (h)

(Vit

alit

y) F

luor

esce

nce

Inte

nsit

y (a

.u.)

MorphologyIn response to dehydration, fronds curled tightly and turned brittle. Such mechanisms is suggested to provide protection against extensive damage.

Relative Water Content (RWC%)RWC is an ideal tool to measure water status of the fronds. In response to the intensity of stress, they adjusted their water status, a mechanism adopted to prevent permanent cell damage.

Glycerol ContentSlow drying (30 C) may induce synthesis / accumulation of glycerol (an osmoprotectant), enabling the fronds to withstand dehydration. However, temperatures ≥ 50 C affected glycerol content, suggesting sensitivity to heat.

VitalitySlow dried (30 C) fronds were metabolically active (alive) even after 72 h of dehydration. However, intermediate (50 C) and fast (80 C) dried fronds were metabolically inactive, suggesting they were dead.

Discussion

Slow dried Polypodium can tolerate dehydration; not high temperature. Osmoprotectant glycerol is one of the component observed to be accumulated 12 h post dehydration, that allows fronds to withstand longer durations of dehydration.

Conclusion

Using RNA seq (gene exprssion), we will determine the time period when different components will be synthesized in a dehydrating frond. Such information will provide deeper understanding on how Polypodiums remain dry for longer durations without dying.

Future Plans

Advisor: Dr. Karl H. Hasenstein

Assembling personal video : Robyn Noltingfor Sigma Xi showcase

Acknowledgements