Arabidopsis NDL-AGB1 modules Play Role in Abiotic Stress and Hormonal Responses Along with Their Specific Functions

Abstract

Arabidopsis N-MYC Downregulated Like Proteins (NDLs) are interacting partners of G-Protein core components. Animal homologs of the gene family N-myc downstream regulated gene (NDRG) has been found to be induced during hypoxia, DNA damage, in presence of reducing agent, increased intracellular calcium level and in response to metal ions like nickel and cobalt, which indicates the involvement of the gene family during stress responses. Arabidopsis NDL gene family contains three homologs NDL1, NDL2 and NDL3 which share up to 75% identity at protein level. Previous studies on NDL proteins involved detailed characterization of the role of NDL1; roles of other two members were also established in root and shoot development using miRNA knockdown approach. Role of entire family in development has been established but specific functions of NDL2 and NDL3 if any are still unknown. Our in-silico analysis of NDLs promoters reveled that all three members share some common and some specific transcription factors (TFs) binding sites, hinting towards their common as well as specific functions. Based on promoter elements characteristics, present study was designed to carry out comparative analysis of the Arabidopsis NDL family during different stages of plant development, under various abiotic stresses and plant hormonal responses, in order to find out their specific and combined roles in plant growth and development. Developmental analysis using GUS fusion revealed specific localization/expression during different stages of development for all three family members. Stress analysis after treatment with various hormonal and abiotic stresses showed stress and tissue-specific differential expression patterns for all three NDL members. All three NDL members were collectively showed role in dehydration stress along with specific responses to various treatments. Their specific expression patterns were affected by presence of interacting partner the Arabidopsis heterotrimeric G-protein β subunit 1 (AGB1). The present study will improve our understanding of the possible molecular mechanisms of action of the independent NDL-AGB1 modules during stress and hormonal responses. These findings also suggest potential use of this knowledge for crop improvement.

Keywords: Arabidopsis; GUS staining; N-Myc downregulated like; PEG; abiotic stress; mannitol; plant hormones; promoter elements.

Figure 1

Mapping of the Arabidopsis N-MYC Downregulated Like Proteins (AtNDL) gene family and putative abiotic stress interacting partners, NDLs stress related interactors (NSRI) on Arabidopsis genome along with the stress related elements in the promoter region of AtNDL gene family. (A) Physical Map of AGI at TAIR using Chromosome Map tool to indicate Chromosomal location of NDLs (NDL1, NDL2, and NDL3) and their stress related interactors in Arabidopsis. The names of the genes are shown to the right of each chromosome and location of genes is indicating their absolute location on respective chromosome. RGS1-Regulator of G protein signaling, ANN1-ANNEXIN, SLTI-Sodium and Lithium-Tolerant 1, RS27A- Arabidopsis Ribosomal Protein, OAS-TL-O-Acetylserine (Thiol) Lyase. (B) In silico analysis of the promoter region of AtNDL gene family promoter regions analyzed to get information about common cis-regulatory elements present on each of the member. (C) Specific cis-regulatory elements present on each AtNDL member. Different colors are used to indicate for each cis-regulatory element.

Figure 2

In silico expression analysis of NDL gene family. (A) GENEVESTIGATOR microarray expression analysis of NDL1, NDL2 and NDL3 during different developmental stages of plant growth. (B) In silico microarray analysis showing tissue-specific expression of NDL1, NDL2, and NDL3 using GENEVESTIGATOR.

Figure 3

In planta expression analysis of NDL gene family during different developmental stagesin presence and absence of AGB1: (A–C, G–I and M–O). Histochemical GUS staining of pNDL1-NDL1-GUS, pNDL2-GUS and pNDL3-GUS seedlings in wild type Col-0 background. (D–F, J–L and P–R) Histochemical GUS staining of pNDL1-NDL1-GUS, pNDL2-GUS and pNDL3-GUS seedlings in agb1-2 mutant background. Histochemical GUS analysis in transgenic Arabidopsis plants during different stages of development was carried out 4 day, 8 day, and 12 day old seedlings were analyzed. GUS expression is under the control of NDL2 and NDL3 promoter and translational fusion in case of NDL1, Scale Bar = 0.2 μM.

Figure 4

In silico expression analysis of NDL gene family during abiotic stress and hormonal treatment using genevestigator and eFP browser: (A). Comparative in-silico expression analysis of NDL1, NDL2, and NDL3 using GENEVESTIGATOR microarray data, under different abiotic stress and hormonal treatments (B). In silico analysis of NDL1, NDL2, and NDL3 during abiotic stress treatments at different time points (0.5, 3, 6, 12, and 24 h) comparative expression is shown as fold change compared to no stress control.

Figure 5

In vivo histochemical GUS activity for NDL1 in wild type background in response to different abiotic stresses and hormones: (A). Histochemical analysis of GUS activity for NDL1 in wild type background in response to different abiotic stress and hormones. Changes in intensity of GUS staining were detected with Heat, Cold, IA, JA, SA and BAP. GUS staining was done in seedlings that were six days old and 24 h of stress treatment followed. (B) Histochemical GUS staining of 6-D-old pNDL1-NDL1-GUS seedlings in wild type Col-0 background. Increased levels of GUS staining intensity detected with PEG at root tip (inset). Result shown is representative of three independent biological replicates (n ≥ 10 in each experiment). Scale Bar = 0.2 μM for seedlings and 0.02 µM for inset RAM.

Figure 6

In vivo histochemical analysis of GUS activity for NDL2 in wild type Col-0 background in response to different abiotic stresses and hormones: Histochemical GUS staining of 6-D-old transgenic Arabidopsis seedlings (pNDL2-GUS) in wild type Col-0 background. Increased intensity levels of GUS staining detected with ABA, cold, and JA. Result shown is representative of three independent biological replicates (n ≥ 10 in each experiment). Scale Bar = 0.2 μM.

Figure 7

In vivo histochemical analysis of GUS activity for NDL3 in wild type Col-0 background in response to different abiotic stresses and hormones: (A). Histochemical GUS staining of 6-D-old transgenic Arabidopsis seedlings (pNDL3-GUS) in wild type Col-0 background. Changes in intensity of GUS staining detected with PEG and Mannitol in RAM. (B) and in hypocotyl part of the seedlings. Result shown is representative of three independent biological replicates (n ≥ 10 in each experiment). Scale Bar = 0.2μM for seedlings and 0.02 μM for RAM.

Figure 8

In vivo histochemical GUS staining of 6-day old transgenic Arabidopsis seedlings (pNDL1-NDL1-GUS) in agb1-2 mutant background: Histochemical GUS staining was done in transgenic Arabidopsis seedlings that were six days old treatment was given for 24 h. (A) Changes in GUS intensity levels were detected with GA, heat and JA. (B). Increased GUS staining detected for Mannitol and PEG at the cell division and elongation part of the primary root. (C) Abiotic stress treatments followed by fluorometric MUG assay was done for quantitative estimation of stress response. Significantly higher GUS activity was obtained for Mannitol and PEG treatment compared to No-stress control indicative of NDL1 involvement during osmotic and drought responses (Students t-test = * p value < 0.05, Error bars represent SD). Result shown is representative of three independent biological replicates (n ≥ 10 in each experiment). Scale Bar = 0.2 μM.

Figure 9

In vivo histochemical GUS staining of 6-day old transgenic Arabidopsis seedlings (pNDL3-GUS) in agb1-2 mutant background: (A). Increased levels of GUS staining intensity were detected in RAM compare to almost no staining in no treatment control with Mannitol and PEG treatment. (B) Histochemical GUS staining was analysed in whole seedling also. ne Six days old Arabidopsis transgenic seedlings were subjected to treatment for 24 h. Result shown is representative of three independent biological replicates (n ≥ 10 in each experiment). Scale Bar = 0.2 μM for seedlings and 0.02 μM for RAM.

Figure 10

AtNDLs responses to various abiotic and hormonal treatments in presence and absence of AGB1: (A) Diagrammatic representation of AtNDLs in wild type background in response to various abiotic and hormonal treatments. (B) AtNDLs responses to various abiotic and hormonal treatments in agb1-2 mutant background. The green lines indicates that the gene was up regulated while the red lines indicates the downregulation of gene after the respective treatments.