Drying without dying- Resurrection fern, Pleopeltis polypodioides

Susan JohnAdvisor: Karl H. Hasenstein

Department of BiologySigma Xi Research Showcase 2016

Hydrated Water stress

Plants are sensitive to water stress

In response to water stress, plants wilt and eventually die!!!No matter how much water is provided to a dead plant, they never come back

BACKGROUND

Water stress is a global problem!!!

In response to water stress crop growth and productivity are significantly lowered, which has resulted in shortage of food supply for ever increasing human population.

In nature there are special group of plants can naturally tolerate water stress known as – ‘Desiccation Tolerant’

If we can identify essential components that make these plants drought tolerant, we can utilize that information to design drought resistant crops, which can be solution to the problem of low food supply.

With the aim of identifying these components, I study Pleopeltis polypodioides (Resurrection fern) because it has not been studied well and it can survive after losing > 95% cellular

water.

RESEARCH HYPOTHESIS

To understand how Pleopletis withstand water stress I explore different characteristics such as -1. Morphology2. Physiology and Biochemistry3. Gene expressions (RNA – Seq)

MORPHOLOGICAL FEATURES

Presence of microstructures has a huge impact on the overall functioning of Pleopeltis

The fronds curl tightly in response to shortage of water and re-expand as soon as water (rain) becomes available. Notice dehydrated fronds are folded such that the abaxial (dorsal) surface of the frond are exposed and adaxial (ventral) surface are curled inward.

Dehydrated fronds Rehydrated (resurrected)

Pleopeltis polypodioides – Resurrection Fern

Sorus

SEM image of peltate scales (150 µm) (trichomes) on abaxial surface of pinna

1 cm

Abaxial (Dorsal) surface of Pleopeltis blade have sori,

stomates, and peltate scales

Close – up view of Pleopeltis(Ventral and Dorsal surfaces of the frond)

Top surface of the scale is known as “SHIELD”

Glabrous adaxial (Ventral) surface of Pleopeltis blade

(Absence of stomates)

1 cm

SEM image of stomates on abaxial surface of pinna

Blade

Pinna

Stipe

Multicellular stalk of the

scale

Vascular bundle

100 µm

The shield of the scale is supported by dome cells and a multicellular stalk that is embedded in epidermis (2 µm section). Peltate scales are a type of trichome. Trichomes are appendages on plants with diverse structure and function.

Transverse Section of Pleopeltis Pinnademonstrate peltate scales are trichome

Shield

ADAXIAL SIDE

ABAXIAL SIDE

Dome cells

Water uptake by fronds in presence and absence of scales

Water uptake in intact fronds increases with time; in absence of the scales the uptake decreases after 1 h of imbibition.

Intact fronds (with scales) take 15 h to lose 50 % loss of cellular water content. However, after removing the scales, descaled fronds require 4 h. Thus, the rate

water loss from descaled frond is significantly faster than intact fronds.

Water loss from fronds in the presence and absence of scales

50 % water loss (Partially Dry)

Dry

Metabolic activity of intact and descaled fronds(measured by reduction of resazurin)

Metabolic activity of the intact fronds decreases gradually over 72 h of dehydration; in the absence of the scales the metabolic activity of the fern declines rapidly

Upon hydrating 72 h dehydrated intact and descaled fronds, the vitality of descaled fronds was significantly reduced compared to intact fronds

BIOCEHMICAL STUDIES

Quantification of different biomolecules in response to dehydration

Fatty Acid measurements from dehydrating fronds

Unsaturated fatty acids such as lineloic acid increased upon dehydration along with saturated fatty acids such as palmitic and stearic acids. However, saturated fatty acids such as eicosanoic and docosanoic acids decreased upon dehydration. These fatty acids may be influencing the membrane integrity of the fronds in response to dehydration.

Reactive Oxygen Species (ROS) and Lipid Hydroperoxide content (LOOH)

Reactive oxygen species (ROS) content increased upto 60 h of dehydration and then declined.

Lipid hydroperoxide (LOOH) content decreased in response to dehydration

ROS and LOOH measurements help understanding the extent of damage to the membrane by free radicals generated in response to water stress. The ROS and LOOH contents were measured by FOX 1 and 2 assays, respectively.

GENE EXPRESSION STUDIES

Highly upregulated transcripts in response to dehydration were determined

Pipeline for RNA – Seq Analysis

Total RNA extracted from hydrated, partially hydrated (50 % RWC) and dehydrated Pleopeltis frondsN = 3 replicates per condition

RNA quality with RIN =7.0 was selected for sequencing on Illumina NGS 500

Over 200 million reads (100 bp) generated

No reference genome available therefore, reads de novo assembled on Trinity

357,785 unique contigs generated (av. Length = 1323 bp)

Contigs blasted against 4 databasesNrplantSwissProtTrEmblKOG(Pleopeltis showed maximum hits with Physcomitrella and Selaginella that are desiccation tolerant moss and lycophyte respectively)

Blast generated 152,095 annotated

contigs

Selected 45,000 that were significantly expressed under

fresh, partially dry and dry conditions

1 2

5

43

67

8

Venn Diagram representing contigs from Fresh, Partially Dry (50 % RWC) and Dry fronds

1079

Fresh

718

Partially Dry

756

Dry

962643

740

8

P – values (significance) of contigs < 0.01

Highly upregulated transcripts and associated contigs were determined by Gene Ontology

Enrichment Analysis (AgriGO 1.2)

Gene Ontology AnnotationsFresh Partially Dry Dry

Summary

• Morphological studies suggest that microstructures such as peltate scales function in water uptake and prevent rapid loss of water, which are necessary for desiccation tolerant plants to gain tolerance.• From transcriptomics studies we were able to determine the

most significant transcripts that were upregulated when the fronds dehydrated.• Our results from Biochemical studies correlate with

transcriptomics studies, which suggest that processes associated with fatty acid metabolism and oxidoreductive activity may be some of the key players functioning in providing tolerance to this plant.

I am now working on identifying potential genes related to fatty acid metabolism, carbohydrate metabolism, oxidoreductive activity and few others and verify them using RT – qPCR.

Ongoing Research…

Acknowledgements

Advisor: Dr. Karl H. Hasenstein

Collaborators: Samuel Roberts Nobel Foundation, Inc. OK

THANK YOU!!