REGULATION OF PIP2-7 AND LATERAL ROOT GROWTH AS A RESPONSE TO DROUGHT IN ARABIDOPSIS

Luke Nelson

BackgroundArabidopsis as a model organism

-quick germination (3-6 days) and flowering (~22 days) (Kumar et. al., 2015)-genome size of 135 Mbs (Balsamo et. al., 2015)-shared responses to many commercial crops such as rice and corn (Valentim et. al., 2015)

Background Aquaporins-water transfer proteins that regulate water transfer and

retention-highly observed in plants such as rice, corn, and Arabidopsis (Qin et. al., 2014)

-Literature suggests that the aquaporin PIP2-7 (Plasma membrane Intrinsic Protein)in Arabidopsis is highly variable in expression when encountering drought conditions (Li et. al., 2016) gifs.co

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Questions Does the water stressed Arabidopsis

respond to drought conditions by laterally growing it’s roots as well as up-regulating aquaporin PIP2-7 expression?

Is there an optimal amount of water consumption for lateral root growth?

Arabidopsis Biological Resource Center

Hypothesis If Arabidopsis is subjected to drought

conditions, then it will respond by laterally growing its roots and expressing

PIP2-7 aquaporin proteins in higher concentrations because of an increased necessity for osmotic balance and water conservation.

Methods1. Stratify seeds in 5*C fridge for 5 days to break dormancy2. Plant seeds in soil and set in 12hr photoperiod/ 25*C

environment 3. After germination period, allow experimental plants to be

reduced to 25, 22, 20, 18, 16, 14, 12, and 10mL water consumption

4. Dissect apical meristem in control and experimental groups, apply methanol and fixative. Degrade cell wall w/ cellulase and wash w/ MTSB and blocking buffer (BSA in PBS)

5. Apply PIP2-7 1* antibody, MTSB, and fluorescent 2* antibody followed by DAPI, Paclitaxel, and Phalloidin in PBS (Pasternak et. al., 2015).

6. Observe under microscope. Record concentration and number of PIP2-7 aquaporins in both groups.

Lateral Root Growth

Results

8 10 12 14 16 18 20 22 24 260

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Water Consumption (mL)

Late

ral

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D.

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Fig. 1. As water consumption increases to 25mL/day, lateral root growth flourishes. Under drought conditions (A.) [10-15mL/day], lateral root growth in A. thaliana ranges from 1.2 – 3.1cm in length. At 22-25mL/day (D.), lateral root length ranges from 7.3 – 9.2cm, suggesting an improvement in root growth in response to normal water consumption.

PIP2-7 Regulation

Fig. 2. Illumination of the secondary PIP2-7 antibody fluoresces when contacting the apical meristem of the control (25mL/day) group. The highly observable aquaporins suggests an up-regulation with increased water consumption.

Fig. 3. There is no observable secondary antibody exhibiting fluorescence. This may be due to inadequate blocking buffer or the down-regulation of PIP2-7 aquaporins in response to drought stress of 10mL/day.

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PIP2-7 Regulation

Fig. 4. The 25mL/day group exhibits pockets of expressed PIP2-7 aquaporins both in lateral root and extracellular space because of mechanical disruption.

Fig. 5. The drought group (10mL/day) expresses a decreased profile of PIP2-7 aquaporins

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PIP2-7 Regulation

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Fig. 6. The apical meristem of a control group plant presents an up-regulated display of PIP2-7

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Fig. 7. The apical meristem of a drought induced plant presents a down-regulated display of PIP2-7

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Results

1 2 3 4 5 6 7 8 9 100

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Observed PIP2-7 Aquaporins

Water Consumption

mL

PIP2-7 Secondary Antibodies

Fig. 8. As water consumption rests at 10mL/day, observed PIP2-7 secondary antibodies ranges from 0-3 fluorophores /250um of plant tissue and 25mL/day ranges from 3-9 fluorophores/250um of plant tissue

Discussion The ability for Arabidopsis

to respond to drought conditions by growing its roots is minimal and suggests an increased reliance on abiotic factors for survival

The shared behavior between commercial crops and Arabidopsis suggests that a multitude of plants will not have sufficient genetic regulation in order to combat/adapt to such

extreme levels of water deprivation.

wbur.org

DiscussionThanks to

-Jeff Duerr’s Lab for use of confocal microscope and PBS, BSAxPBS, Paclitaxel, and Phalloidin

-Joanna Porankiewicz-Asplund from Agrisera for PIP2-7 Plasma membrane aquaporin isoforms 1-7 (C-terminal) Primary Antibody and Goat anti-Rabbit IgG (H&L) conj. Secondary Antibody

Literature CitedBalsamo, R., M. Boak , K. Nagle, B. Peethambaran, & B. Layton. 2015. Leaf biomechanical properties in arabidopsis thaliana

polysaccharide mutants affect drought survival. Journal of Biomechanics 48:4124-4129.

Kumar M.N., H. Yi-Fang, and V. Paul E. 2015. At14A-like1 participates in membrane-associated mechanisms promoting growth during

drought in arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 112: 10545-

50.

Li, R., J. Wang, S. Li, L. Zhang, C. Qi, Weeda, Y. Guo. 2016. Plasma membrane intrinsic proteins SlPIP2;1, SlPIP2;7 and SlPIP2;5

conferring enhanced drought stress tolerance in tomato. Scientific Reports 302:2045-2322.

Pasternak, T., O. Tietz, K. Rapp, M. Begheldo, R. Nitschke, B. Ruperti, K. Palme. 2015. Protocol: an improved and universal procedure

for whole-mount immunolocalization in plants. Plant Methods 12:1150-1186.

Qin, L., Y. Li, D. Li, W. Xu, Y. Zheng, & X. Li. 2014. Arabidopsis drought-induced protein Di19-3 participates in plant response to

drought and high salinity stresses. Plant Molecular Biology 86:609-625.

Valentim F.L., S. Van Mourik, S. Pose, D. Kim, C. Min, M. Schmid, R. Van Ham, J. Roeland, M. Busscher, G. Sanchez-Perez, J.

Molenaar, G. Angenent, R. Immink, and A. Van Dijk. 2015. A quantitative and dynamic model of the arabidopsis flowering time

gene regulatory network. Plos One 10:1932-6203.