Childhood memories of summer vacation involved watering the plants and taking care of the parent’s garden. It was filled with fun and intrigue as a new shoot with small leaves would sprout from a potato bud, or, out of nowhere a ”Touch me not” plant would grow, and when touched its leaves would shrink. Plants respond to stimuli like touch, light, water, soil, and have necessary adaptations to them.
Henceforth, these kitchen gardens would have seen a lot of experimentation. How a plant grew under different lights (red, blue, and white) or how they respond in the soil and in a pot or if they grew tall for piano or guitar music were some of the classic experiments. And all these experiments proved that plants respond to stimuli faster and some (especially 80’s rock n roll) helped them grow well.
De-etiolation of plants
One such process is how seeds respond to light. Seeds undergo a process called etiolation when not exposed to light, i.e., they promote longer hypocotyls (to be stem) growth to reach soil surface soon, and have closed cotyledons that lack chlorophyll. They de-etiolate when they are first exposed to light. This exposure causes the plant to grow chloroplast, develop cotyledons, and reduce the speed of hypocotyls growth.
This response of plants poses a question, what is the molecular basis of this differential response to the availability of light? Many researches have found transcription factors and genes responsible for these stimuli. A recent paper by Burko, Yogev, et al(1) discusses the role of transcription factor HY5 in the de-etiolation process of plants. The HY5 transcription factor regulates gene expression and some of the primary genes can be found by the RNA-Seq data from thale cress (Arabidopsis thaliana).
To understand the role of HY5, plant mutants with no HY5 (hy5), a silencing factor of HY5 (HY5-SRDX), an activator of HY5 (HY5-VP16), native type (HY5-OX) and control-Columbia 0 (Col) were sequenced immediately as they were exposed to light. The sequence files were aligned and quantified. This data was used to check if HY5 regulates the de-etiolation process for experimentation shifting from backyard garden to a laptop.
Null Hypothesis: HY5 regulates the de-etiolation process
Interpreting the transcripted transcription factor
A new RNA analysis experiment was created in Strand NGS using the quantified files. Once the experiment was created, the samples were grouped according to their types and interpretations created. This labeling plant is from the paper(1) to identify which group they belong to. Once labeled we can clearly differentiate on how they respond to their stimuli.
Once the experiment is created we can visualize the gene expression of all genes in all samples by various plots.
The scatter plot illustrates the mean and difference of gene expression between 2 samples. It gives a view as to how scattered the genes are in 2 samples
Box plot illustrates a box-whisker plot of gene expression values in each sample.
The profile plot illustrates the profile of expression values for samples based on grouping we give.
In the garden, the responses were measured from morphological characteristics as the stem length, the color of the leaf, which plant flowered first (also called the phenotype changes). Genotype changes can be seen through the change in gene expression. Using the grouping, the differential gene expression between these HY5 groups can be found out.
Differential expression was analyzed by ANOVA (in Replicate Analysis) and Benjamini Hochberg correction, with a p-value cutoff of ≤0.05 and fold change ≥1 with respect to no HY5 mutant (hy5). 6561 genes were found to be differentially expressed.
These genes are expressed when HY5 is expressed. Silencing domains in HY5-SRDX ideally silences the genes that bind to them and activating domains in HY5-VP16 activates the gene. So, a gene that binds to HY5 will be downregulated in HY5-SRDX and upregulated in HY5-VP16. This mechanism reduces false positives. So, the differential expression of HY5-SRDX and HY5-VP16 was done to see genes that do not have this effect. 820 genes matched this criterion.
Can the samples be clustered based on these 820 genes? A PCA and hierarchical clustering were done based on these 820 genes on the plants exposed to light
The PCA plot clearly separated the 5 samples into separate clusters. And the HY5-SRDX samples are far from HY5-VP16 samples. Also, it could be seen HY5-Col and HY5-OX samples are seen near as they are native forms of HY5. Also, they have nearly the same Principal component-1 value as that of HY5-VP16. These are plants that express HY5. hy5 is near HY5-SRDX and does not express HY5 and HY5-SRDX silences the genes.
Hierarchical clustering also clusters samples the same way as PCA. Hence it is clear these 820 genes are expressed by HY5.
So, now what do these 820 genes do? GO-Analysis was run to check that. The top GO processes identified are in the table below.
The GO terms clearly indicate response to stimuli. Hence, it can be concluded that HY5 helps in the de-etiolation process of plants.
Null Hypothesis accepted.
And it was fun to recreate and analyze an experiment in-silico to understand how HY5 helps in plant response. Now, you also can analyze your garden observations using Strand NGS as described.
- Burko, Yogev, et al. “Chimeric activators and repressors define HY5 activity and reveal a light-regulated feedback mechanism.” The Plant Cell 32.4 (2020): 967-983.