Document Type

Restricted

Advisor

Rachel Spicer

Publication Date

2023

Comments

This paper is restricted to the Connecticut College campus until May 22, 2024.

Abstract

As drought becomes more pronounced due to climate change, it is important to characterize the responses of plants to water stress. One area of interest includes how auxin, the hormone most associated with growth and development, is affected by drought. In particular, the final size of an expanding leaf is often reduced in response to drought and auxin is known to play a key role in leaf expansion. The YUCCA proteins catalyze the conversion of indole-3-pyruvic acid to indole-3-acetic acid (IAA), the dominant form of auxin in most plants, and most of this IAA is produced in rapidly expanding leaves. However, in addition to synthesizing IAA, the YUCCA proteins are also associated with thiol reductase activity. This enzymatic function decreases the amount of reactive oxygen species (ROS), which can accumulate in plant tissue under stress such as drought. The YUCCA gene family in Populus includes 12 YUCCAs, but they are not well described. This experiment was aimed to assess whether YUCCA genes in Populus leaves at different stages of development are differentially expressed in response to drought. Multi-shoot, one-year-old hybrid poplar plants (Populus tremula x alba; INRA 717-1B4) were either watered to field capacity daily (control; n = 5) or deprived of water (drought; n = 5) for nine days. Over that period, stomatal conductance, and stem water potential both decreased significantly in the droughted trees relative to the control trees. The shoot apex, leaf 8, and leaf 16 (i.e., the eighth and sixteenth leaves counted down from the apex) were harvested for gene expression analysis, with the apex representing rapid growth, leaf 8 representing a leaf approximately one half the size of the average fully mature leaf, and leaf 16 representing a fully expanded leaf. Leaf relative water content (RWC) measured on leaf disks at the time of harvest and showed that the RWC of leaf 8 was significantly reduced under drought compared to controls, but the RWC of leaf 16 was similar between treatments. qRT-PCR was used to determine expression levels of five different YUCCA genes that had previously been shown to be expressed in Populus leaves: YUC1, YUC2, YUC4, YUC6, and YUC12. These results were normalized using actin, ubiquitin, and tubulin, genes that were found to significantly decrease in their expression in the droughted plants when their Cq values were used to take a geometric mean. Results were then analyzed by normalizing with this geometric mean as well as with the gene least affect by drought, ubiquitin. It was shown that YUC12 did not significantly change between treatments in the apices in either method of normalization. However, YUC1, YUC4, and YUC6 had decreased expression in drought in the apices, as well as in some of the leaf samples when normalized with ubiquitin. YUC2 was initially normalized to have significantly increased expression in the droughted plants, however this result was made insignificant by normalization with ubiquitin. Future work should find stable normalizer genes to draw more reliable conclusions from as well as measure IAA concentrations in leaf tissue under drought to better understand these hormonal cues.

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The views expressed in this paper are solely those of the author.