Uncovering the molecular signature underlying the light intensity-dependent root development in Arabidopsis thaliana

Abstract

Background: Root morphology is known to be affected by light quality, quantity and direction. Light signal is perceived at the shoot, translocated to roots through vasculature and further modulates the root development. Photoreceptors are differentially expressed in both shoot and root cells. The light irradiation to the root affects shoot morphology as well as whole plant development. The current work aims to understand the white light intensity dependent changes in root patterning and correlate that with the global gene expression profile.

Results: Different fluence of white light (WL) regulate overall root development via modulating the expression of a specific set of genes. Phytochrome A deficient Arabidopsis thaliana (phyA-211) showed shorter primary root compared to phytochrome B deficient (phyB-9) and wild type (WT) seedlings at a lower light intensity. However, at higher intensity, both mutants showed shorter primary root in comparison to WT. The lateral root number was observed to be lowest in phyA-211 at intensities of 38 and 75 μmol m ^{- 2} s ^{- 1}. The number of adventitious roots was significantly lower in phyA-211 as compared to WT and phyB-9 under all light intensities tested. With the root phenotypic data, microarray was performed for four different intensities of WL light in WT. Here, we identified ~ 5243 differentially expressed genes (DEGs) under all light intensities. Gene ontology-based analysis indicated that different intensities of WL predominantly affect a subset of genes having catalytic activity and localized to the cytoplasm and membrane. Furthermore, when root is irradiated with different intensities of WL, several key genes involved in hormone, light signaling and clock-regulated pathways are differentially expressed.

Conclusion: Using genome wide microarray-based approach, we have identified candidate genes in Arabidopsis root that responded to the changes in light intensities. Alteration in expression of genes such as PIF4, COL9, EPR1, CIP1, ARF18, ARR6, SAUR9, TOC1 etc. which are involved in light, hormone and clock pathway was validated by qRT-PCR. This indicates their potential role in light intensity mediated root development.

Keywords: Auxin; Gene expression; Hormone; Intensity; Light signaling; Root.

Fig. 1

Light intensity dependent root growth varies in phytochrome mutants. a Phenotypic differences in root architecture. b Primary root length (mm) for WT, phyB-9 and phyA-211 under four different WL intensities of 38, 75, 112 and 150 μmol m ^− 2 s ^− 1. The analysis was done in 6-days old seedlings. Error bars represent SE. The means were compared using Tukey test with p ≤ 0.05. Same letters denote statistical significance. There were five technical replicates, each replicate consists of 20 seedlings. Scale bar = 10 mm

Fig. 2

Phytochrome mutants show variability in light intensity dependent lateral and adventitious root growth. a Lateral and adventitious root architectural differences. The lateral and adventitious root have been indicated by arrowheads and arrows respectively. b Phytochrome mutants show variation in lateral root number. c Adventitious root number varies among the genotypes under all four WL intensity. The analysis has been done in 6-days old seedlings. Error bars represent SE. The means were compared using Tukey test with p ≤ 0.05. Same letters denote statistical significance There were three technical replicates, each replicate consists of 20 seedlings. Scale bar is 10 mm

Fig. 3

DEGs found under different comparative light intensities from microarray. Total number of DEGs, number of upregulated and downregulated DEGs under comparative light intensity of 150 vs 112, 150 vs 75, 150 vs 38, 112 vs 75, 112 vs 38 and 75 vs 38 μmol m ^− 2 s ^− 1 obtained from 5-days old WT root microarray analysis

Fig. 4

Venn diagram representation of overlapping and differential DEGs under variable white light intensity. a DEGs for 150 vs 112, 150 vs 75 and 150 vs 38 μmol m ^− 2 s ^− 1. b DEGs for 150 vs 112, 112 vs 75 and 112 vs 38 μmol m ^− 2 s ^− 1. c DEGs for 150 vs 75, 112 vs 75 and 75 vs 38 μmol m ^− 2 s ^− 1. d DEGs for 150 vs 38, 112 vs 38 and 75 vs 38 μmol m ^− 2 s ^− 1 light intensity from microarray analysis of WT root

Fig. 5

Classification of DEGs under different categories and terms of GO annotation analysis. a DEGs under 150 vs 112 μmol m ^− 2 s ^− 1 b DEGs under 150 vs 75 μmol m ^− 2 s ^− 1 c DEGs under 150 vs 38 μmol m ^− 2 s ^− 1 d DEGs under 112 vs 75 μmol m^{− 2} s ^− 1 e DEGs under 112 vs 38 μmol m ^− 2 s ^− 1 f DEGs under 75 vs 38 μmol m^− 2 s^− 1 light intensity from microarray analysis for the functionality of DEGs in 5-days old WT root

Fig. 6

Genes involved in light, hormone and clock-regulated pathways are differentially expressed under 150 vs 112 μmol m ^− 2 s ^− 1 light intensity. Light signaling genes (1a) COL3, (1b) EPR1 and (6.2c) PIF4 showed differential regulation; (2a) ARF4 and (2b) ARF2 genes involved in auxin signaling were differentially expressed; (3) TOC1 gene showed variability in its expression in case of WT; (4a) ARR6 and (4b) CIP1 genes were differentially expressed in Ler, 35S::PhyBGFP, phyB-5 and phyB-5 respectively under 150 vs 112 μmol m ^− 2 s ^− 1 light intensity. qRT-PCR was performed with root samples of 5-days old seedlings. Analysis has been described in the method section

Fig. 7

Relative expression of genes involved in light, hormone and clock-regulated pathways is influenced under 150 vs 75 μmol m ^− 2 s ^− 1 light intensity. Differential expression of (1a) CSN6B, (1b) EPR1, (1c) PIF4 and (1d)COL3 genes; Variation in gene expression of (2a) ARF18, (2b) LAX2, (2c) CIP1 and (2d) KMD1 ; (3) TOC1 gene regulation in WT; (4a) ARR6 showed variation in its regulation in all genotypes but (4b) CIP1 expression varied only in case of Ler and phyB-5 under 150 vs 75 μmol m ^− 2 s ^− 1 light intensity. qRT-PCR was performed with root samples of 5-days old seedlings. Analysis has been described in the method section

Fig. 8

Light intensity of 150 vs 38 μmol m ^− 2 s ^− 1 affects the expression profile of genes involved in light, hormone and clock-regulated pathways. Variation in expression of (1a) COL9, (1b) EPR1 and (1c) PIF4 candidate genes; (2a) CIP1, (2b) ARF18, (8.2c) ARR6, (2d) SAUR9 and (2e) LAX2 genes showed variable expression; (3) Regulation in TOC1 gene expression in WT root; (4a) ARR6 expression varied in all genotypes but (4b) CIP1 expression varied only in case of Ler and phyB-5 under 150 vs 38 μmol m ^− 2 s ^− 1 light intensity. qRT-PCR was performed with root samples of 5-days old seedlings. Analysis has been described in the method section

Fig. 9

Relative expression of genes involved in light signaling pathways under 112 vs 75 μmol m ^− 2 s ^− 1 light. Gene expression profiling of (1a) EPR1 and (1b) COL3 varied in WT root; Expression of (2a) ARR6 varied in all seed lines and (2b) CIP1 was differentially expressed in 35S::PhyBGFP and phyB-5 only under 112 vs 75 μmol m ^− 2 s ^− 1 light intensity. The qRT-PCR was performed with root samples of 5-days old seedlings. Analysis has been described in the method section

Fig. 10

Expression profiling of genes involved in light, hormone and clock-regulated pathways is influenced under 112 vs 38 μmol m ^− 2 s ^− 1 light intensity. (1a) COL3 and (1b) EPR1 genes were differentially expressed; Variation in expression profile of (2a) ARF2, (2b) SAUR9, (2c) SAUR26 and (2d) ARR6 genes; (3) CCA1 gene expression varied in WT; Expression profiling of (4a) ARR6 and (4b) CIP1 genes varied in all seed lines under 112 vs 38 μmol m ^− 2 s ^− 1 light intensity. qRT-PCR was performed with root samples of 5-days old seedlings. Analysis has been described in the method section

Fig. 11

Change in relative expression of genes involved in light, hormone and clock-regulated pathways under 75 vs 38 μmol m ^− 2 s ^− 1 light intensity. Gene expression profile of (1a) CSN6A, (1b) CSN6B, (1c) EPR1, (1d) HY5 and (1e) COL9  varied; (2a) ARF18, (2b) CIP1, (2c) KMD1 and (2d) LAX2 genes showed variable expression; (3) CCA1 gene showed variable expression; Expression profiling of (4a) ARR6 and (4b) CIP1 genes differ in all seed lines under 75 vs 38 μmol m ^− 2 s ^− 1 light intensity. qRT-PCR was performed with root samples of 5-days old seedlings. Analysis has been described in the method section