Transcriptional response to petiole heat girdling in cassava

Abstract

To examine the interactions of starch and sugar metabolism on photosynthesis in cassava, a heat-girdling treatment was applied to petioles of cassava leaves at the end of the light cycle to inhibit starch remobilization during the night. The inhibition of starch remobilization caused significant starch accumulation at the beginning of the light cycle, inhibited photosynthesis, and affected intracellular sugar levels. RNA-seq analysis of heat-treated and control plants revealed significantly decreased expression of genes related to photosynthesis, as well as N-metabolism and chlorophyll biosynthesis. However, expression of genes encoding TCA cycle enzymes and mitochondria electron transport components, and flavonoid biosynthetic pathway enzymes were induced. These studies reveal a dynamic transcriptional response to perturbation of sink demand in a single leaf, and provide useful information for understanding the regulations of cassava under sink or source limitation.

Figure 1. Physiological analysis of the control, partial girdled (PG) and fully girdled (FG) leaves.

(A) An IKI stain showing starch accumulation in control, PG and FG cassava leaf lobes. Leaves were collected from 120- and 45-day-old plants at 0, 2, and 4 h after the lights came on, cleared of photosynthetic pigments, and stained with IKI. (B) Starch and sucrose measurements for control, PG and FG leaves. All leaves were harvested and processed concurrently. Letters above the standard error bars indicate whether the treatment had a significant influence within leaves from plants of a given age (P < 0.05) based on analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) test. (C) A/Ci curve. Pn (A) vs intercellular CO₂ concentration (Ci; μmolmol⁻¹). Measurements were performed at an average ambient relative humidity of 46.6 ± 3.4% and an irradiance of 1200 μmol m⁻²s⁻¹.

Figure 2. A heat map showing under and over represented functional classifications among genes which were differentially regulated in partial girdled (PG) and fully girdled (FG) cassava leaves.

This analysis was performed using the Pageman software package http://mapman.gabipd.org/web. Red boxes indicate that genes in a category were generally down-regulated in the corresponding set of samples relative to controls while blue boxes indicate that genes in a category were generally up-regulated in the corresponding set of samples. Fisher's exact test was applied to test for enrichment of functional category and FDR was controlled for by Benjamini and Hochberg's procedure at P = 0.01.

Figure 3. Effects of girdling on the expression of genes associated with photosynthesis, N metabolism and tetrapyrrole synthesis pathways.

The values in red and green indicate log₂ fold increases and decreases, respectively, in expression of individual genes within each category in treated leaves relative to control leaves.

Figure 4. Effects of girdling on the expression of genes associated with (A) Glycolysis, TCA cycle, and mitochondrial electron transport pathways and (B) flavonoid biosynthetic pathway.

The values in red and green indicate log₂ fold increases and decreases, respectively, in the expression of specific genes in partial girdled (PG) and fully girdled (FG) leaves compared to control leaves. Experimental conditions 1–6 (left to right) are leaves from 120-day-old plants that were developing storage roots: (1) 0 h PG leaves, (2) 0 h FG leaves, (3) 2 h PG leaves, (4) 2 h FG leaves, (5) 4 h PG leaves, and (6) 4 h FG leaves; conditions 7–10 (left to right) are leaves from 45-day-old seedlings that have no storage roots developed: (7) 0 h PG leaves, (8) 0 h FG leaves, (9) 2 h PG leaves, and (10) 2 h FG leaves.

Figure 5. Effects of girdling on the expression of genes associated with starch and sucrose metabolism pathways.

The values in red and green indicate log₂ fold increases and decreases, respectively, in expression in partial girdled (PG) and fully girdled (FG) leaves compared to control leaves. Each row represents the expression pattern of a specific gene. Each column represents a different experimental condition. Experimental conditions 1–6 (left to right) are leaves from 120-day-old plants that were developing storage roots: (1) 0 h PG leaves, (2) 0 h FG leaves, (3) 2 h PG leaves, (4) 2 h FG leaves, (5) 4 h PG leaves, and (6) 4 h FG leaves; conditions 7–10 (left to right) are leaves from 45-day-old seedlings that have no storage roots developed: (7) 0 h PG leaves, (8) 0 h FG leaves, (9) 2 h PG leaves, and (10) 2 h FG leaves.

Figure 6. Effects of girdling on the expression of transcription factors.

For each row, the values in red and green indicate log₂ fold increases and decreases, respectively, in expression of a specific gene in partial girdled (PG) and fully girdled (FG) leaves compared to the control.