Salt stress and senescence: identification of cross-talk regulatory components

Abstract

Leaf senescence is an active process with a pivotal impact on plant productivity. It results from extensive signalling cross-talk coordinating environmental factors with intrinsic age-related mechanisms. Although many studies have shown that leaf senescence is affected by a range of external parameters, knowledge about the regulatory systems that govern the interplay between developmental programmes and environmental stress is still vague. Salinity is one of the most important environmental stresses that promote leaf senescence and thus affect crop yield. Improving salt tolerance by avoiding or delaying senescence under stress will therefore play an important role in maintaining high agricultural productivity. Experimental evidence suggests that hydrogen peroxide (H2O2) functions as a common signalling molecule in both developmental and salt-induced leaf senescence. In this study, microarray-based gene expression profiling on Arabidopsis thaliana plants subjected to long-term salinity stress to induce leaf senescence was performed, together with co-expression network analysis for H2O2-responsive genes that are mutually up-regulated by salt induced- and developmental leaf senescence. Promoter analysis of tightly co-expressed genes led to the identification of seven cis-regulatory motifs, three of which were known previously, namely CACGTGT and AAGTCAA, which are associated with reactive oxygen species (ROS)-responsive genes, and CCGCGT, described as a stress-responsive regulatory motif, while the others, namely ACGCGGT, AGCMGNC, GMCACGT, and TCSTYGACG were not characterized previously. These motifs are proposed to be novel elements involved in the H2O2-mediated control of gene expression during salinity stress-triggered and developmental senescence, acting through upstream transcription factors that bind to these sites.

Keywords: Arabidopsis; hydrogen peroxide; longevity; reactive oxygen species; salt stress; senescence; signal cross-talk; transcription factor..

Fig. 1.

Molecular and physiological analysis of salt-treated plants. (a) Design of the experiment and plant stage selected for salt stress (150mM NaCl) treatment (28 d after sowing, DAS). Samples were collected 6h after the onset of the stress (short-term salinity stress). The later time point (4 d after start of salinity stress) was selected based on the percentage reduction of relative leaf chlorophyll (chl) content. (b) Leaf chlorophyll content. (c) Photochemical efficiency of PSII (F _v/F _m). The values in (b) and (c) represent means of data obtained for 13 plants at each data point ±SD. (d) Quantitative RT-PCR analysis of SAG12 and (e) WRKY53 expression under control (grey column) and salt stress (black column) conditions at two examined time points. Data are means of three independent experiments ±SD. (f) Venn diagram showing an overview of changes in gene expression (>2-fold) of ROS-responsive genes in 6h and 4 d NaCl-treated samples and their shared responses. The numbers in the Venn diagram indicate the number of genes (upper values indicate the number of up-regulated genes, and lower values indicate the number of down-regulated genes).

Fig. 2.

Shared gene expression responses. (a) Venn diagram showing the overlap of genes differentially expressed during developmental and salt-induced senescence. (b) Venn diagram showing the overlap of genes affected during developmental senescence, salt-induced senescence, and upon hydrogen peroxide (H₂O₂) treatment. Numbers indicate the number of genes up-regulated (upper values) or down-regulated (lower values) in the different conditions.

Fig. 3.

Cis-regulatory search pipeline. The analysis pipeline included the following steps. (A) Upstream sequences (500bp) of input genes were retrieved from the TAIR database. (B) MEME Suite was used to identify significantly enriched sequence motifs in the set of queried promoters (here, the CACGTGT motif is given as an example). (C) Candidate motifs were compared with a database of known cis-regulatory motifs using TOMTOM. (D) A list of all Arabidopsis genes containing the motif was retrieved using PatMatch. (E) This gene set was trimmed to include only those genes that were identified in a repeated MEME analysis. (F) Finally, function enrichment analysis was performed using agriGO. Database logos were taken from the respective web pages.

Fig. 4.

Network representation of the correlation structure of genes responsive to long-term moderate salt stress, oxidative stress, and affected during developmental senescence. All network communities are colour-coded; for communities containing more than five genes (1–7), the CREs enriched in the gene’s promoters (with a P-value cut-off of 0.0001) are listed.

Fig. 5.

Model for the integration of H₂O₂ in developmental and salinity-induced senescence. Hydrogen peroxide (H₂O₂) accumulates in leaves during developmental senescence, as reported, for example, by Zimmermann et al. (2006). Salinity stress similarly triggers a rise in cellular H₂O₂ level. Transcription factors (TFs) responding to an elevated H₂O₂ level activate genes included in cluster 26, most probably by binding to cis-regulatory elements (CREs) identified here and by Petrov et al. (2012). Genes contained in cluster 1 may be down-regulated by TFs through unknown CREs. Salinity may additionally elicit stress responses not directly linked to senescence.