Elsevier

Plant Physiology and Biochemistry

Volume 147, February 2020, Pages 235-241
Plant Physiology and Biochemistry

Research article
Nuclease and ribonuclease activities in response to salt stress: Identification of PvRNS3, a T2/S-like ribonuclease induced in common bean radicles by salt stress

https://doi.org/10.1016/j.plaphy.2019.12.016Get rights and content

Highlights

  • The T2 ribonuclease (RNase) family in French bean is composed by 13 members grouped in three classes.

  • Three main RNases are detected in radicles under salt stress, being one of them absent under non stress conditions.

  • PvRNS3, a Class I member, is highly induced in radicles under salt stress.

Abstract

The increase in soil salinization due to global climate change could cause large losses in crop productivity affecting, among other biological processes, to germination and seedling development. We have studied how salt stress affects nucleic acid degrading activities in radicles of common bean during seedling development. In radicles of common bean, a main nuclease of 37 kDa and two ribonucleases of 17 and 19 kDa were detected. Saline stress did not alter these three activities but induced a new ribonuclease of 16 kDa. All three ribonucleases are acidic enzymes that were inhibited by Zn. The 16 and 17 kDa ribonucleases are inhibited by guanilates. In the genome of common bean, we have identified 13 genes belonging to the T2 ribonuclease family and that are grouped in the 3 classes of T2 ribonucleases. The analysis of the expression of the 3 genes belonging to Class I (PvRNS1 to 3) and the unique gene from Class II (PvRNS4) in radicles showed that PvRNS3 is highly induced under salt stress.

Introduction

Salt stress reduces plant growth and is responsible for loss of crops around the Word, being the abiotic stress factor that most severely limit crop productivity (Munns and Tester, 2008). In the next decades, this problem will worsen, since an increase in soil salinization is expected due to climate change (AbdElgawad et al., 2016). An excess of salt causes an ionic imbalance that leads to the formation of toxic ions, generation of osmotic stress, and the appearance of reactive oxygen species (ROS), which damage membrane lipids, nucleic acids and proteins (Chaparzadeh et al., 2004).

Seed germination and post-germinative growth are two crucial processes for the establishment and survival of plants under adverse situations. Salt stress affects both processes, reducing germination rate and the growth of seedling (Pandey and Penna, 2017), which could be due to a reduction in the mobilization of reserves in seeds (Gomes-Filho et al., 2008). Salt tolerance is achieved by several mechanisms at cellular, tissue or whole plant levels. Among them, changes in root and shoot length, accumulation of osmolytes and maintenance of K+/Na+ ratio are the most relevant (Pandey and Penna, 2017). In stress conditions, seedlings should recycle the damaged or useless organelles and compounds in order to maintain cellular homeostasis. This is achieved by a process known as autophagy, which has been proposed to promote stress tolerance (Signorelli et al., 2019).

Nucleic acids are organic compounds relatively abundant in plant tissues. They contain nitrogen and phosphorous, in addition to carbon and, therefore, could have a relevant role in plant physiology as nutrient reservoirs, point that has received little attention. Nucleic acids are typically the largest organic phosphorous pool in plants (Veneklaas et al., 2012). In common bean, activities involved in the degradation of nucleic acids have been described in situations that demand high nutrient mobilization such as embryonic axes development (Lambert et al., 2014) and cotyledons and leaf senescence (Lambert et al., 2016, 2017). In wheat, the degradation of RNA and the catabolism of the nucleotides support the growth in nitrogen starved conditions (Melino et al., 2018). Nucleotides are the monomers that constitute nucleic acids; they act as energy intermediaries, and are precursors of vitamins of class B, essential coenzymes (NAD, FAD and SAM) and secondary metabolites (Zrenner et al., 2006). Nucleotides can be synthesized de novo, from amino acids, or from nitrogenous bases or nucleosides from recycling pathways (Zrenner et al., 2006). The availability of nucleotides in the early post-germinative development of the seedlings is critical (Stasolla et al., 2003), to the extent that the balance between synthesis, recycling and degradation of nucleotides during this stage seems to be crucial to the success of germination (Mohlmann et al., 2010). The demand of nucleotides in seedlings is high to synthesize, among other molecules, their nucleic acids.

Among nucleic acids, RNA is the most abundant molecule in plant cells, representing around 85% of total nucleic acids and 47% of total organic P (Veneklaas et al., 2012). Ribonucleases (RNases) are enzymes that catalyse the degradation of RNA into smaller components. Ribonucleases are ubiquitous components in living cells and participate in a wide variety of processes (Zheng et al., 2014). Due to the relative abundance of ribonucleic acids, ribonuclease activities could play a relevant role in situations of nutrient mobilization. Multiple RNases have been identified which can be grouped in more than 15 superfamilies (Aravind and Koonin, 2001). The most widespread family of ribonucleases is the T2 family with representatives in viruses, bacteria, fungi and eukaryotes (Luhtala and Parker, 2010). Its members are classified into three classes (I to III) (Ramanauskas and Igic, 2017). The genes encoding RNases of class I have been described so far only in plants, and encode enzymes with specialized functions such as regulation of the stress response, defence against microorganisms, phosphate and nitrogen storage (Zheng et al., 2014). In contrast, genes encoding class II RNases, which are conserved among eukaryotes, have a role in the maintenance of cellular homeostasis, and are often constitutively expressed (Zheng et al., 2014). Ribonucleases of class III are components of the system of self-incompatibility, play a key role in the recognition and rejection of pollen, and have not been identified in the genome of Arabidopsis (Ramanauskas and Igic, 2017). T2 RNases are either secreted or targeted to membrane bound compartments of the secretory pathway such as the ER, lysosome, or vacuole (Irie, 1999; MacIntosh, 2011).

Class I and Class II are also known as S-like ribonucleases (Green, 1994) and, although the biological role of these enzymes is poorly understood, it has been suggested to be involved in phosphate recycling in several plant species (Bariola et al., 1994; Liang et al., 2002; Kock et al., 2006; Rojas et al., 2013). Induction of ribonucleases and nucleases in response to salt stress has been described (Bariola et al., 1994; Zheng et al., 2014; Jiang et al., 2008; Sui et al., 2019).

In our laboratory, we are interested in investigating the role of nucleic acid catabolism under situations of nutrient mobilization. In this paper, we present the identification of 13 genes belonging to the ribonuclease T2 family in common bean, and report changes in ribonuclease activity and gene expression in radicles of seedlings exposed to salt stress.

Section snippets

Materials and methods

Common bean seeds (Phaseolus vulgaris L cv. Great Northern) were sterilized and germinated as previously indicated (Lambert et al., 2014). Five days after the start of imbibition, the seeds were transferred to Petri dishes with paper moistened with distilled water (control) or with paper moistened with a solution of 100 and 200 mM sodium chloride, and were maintained for 24 h under the same culture conditions. After this time, the radicle was separated from the rest of the seedling and

Nucleic acid degrading activities in response to salt stress

Nucleic acid degrading activities were assayed in common bean radicles from control and salt treated seedlings using an in-gel assays with different nucleic acid imbibed on the gel during polymerization (Fig. 1). Although the salt treatment reduced the rate of seedling growth, with both salt concentrations the fresh weigh of embryonic axes increased suggesting a moderate effect of the salt in seedling development. No nuclease activities that degrade double-stranded DNA were detected (Fig. 1A).

Discussion

Crop productivity under unfavourable conditions will be a main challenge in future agriculture and saline stress will be one of the most important adverse factors. To cope with these stresses, plants develop several strategies, among which are the recycling of damaged or useless organelles and compounds. The importance of nucleic acids in nutrient remobilization has received little attention despite its possible contribution to the nitrogen and phosphorous pools. In order to investigate the

Author contributions statement

GG-V and PP conceived the research plan and designed the experiments. MD-B, ED-G, GG-V performed the experiments. PP was the primary author involved in writing the original draft of the paper. GG-V and MP contributed to review and editing of the paper. All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors thank Marta Robles for her technical assistance, and Central Service for Research Support (SCAI, Universidad de Córdoba) for sequencing assistance. M Diaz-Baena acknowledges the support of fellowship from Universidad de Córdoba (Beca Semillero) and Ministerio de Educación Cultura y Deporte (Beca de Colaboración). E. Delgado-Garcia acknowledges the support of a contract from Sistema Nacional de Garantía Juvenil and the Programa Operativo de Empleo Juvenil (Junta de Andalucía y Fondo

References (44)

  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method

    Methods

    (2001)
  • N. Luhtala et al.

    T2 Family ribonucleases: ancient enzymes with diverse roles

    Trends Biochem. Sci.

    (2010)
  • M. Pandey et al.

    Time course of physiological, biochemical, and gene expression changes under short-term salt stress in Brassica juncea L

    Crop J

    (2017)
  • C. Stasolla et al.

    Purine and pyrimidine nucleotide metabolism in higher plants

    J. Plant Physiol.

    (2003)
  • W.T. Sui et al.

    Arabidopsis Ca2+-dependent nuclease AtCaN2 plays a negative role in plant responses to salt stress

    Plant Sci.

    (2019)
  • J. Zheng et al.

    Overexpression of an S-like ribonuclease gene, OsRNS4, confers enhanced tolerance to high salinity and hyposensitivity to phytochrome-mediated light signals in rice

    Plant Sci.

    (2014)
  • H. AbdElgawad et al.

    High salinity induces different oxidative stress and antioxidant responses in maize seedlings organs

    Front. Plant Sci.

    (2016)
  • J.J.A. Armenteros et al.

    DeepLoc: prediction of protein subcellular localization using deep learning

    Bioinformatics

    (2017)
  • T. Avin-Wittenberg

    Autophagy and its role in plant abiotic stress management

    Plant Cell Environ.

    (2019)
  • P. Baldrich et al.

    Plant extracellular vesicles contain diverse small RNA species and are enriched in 10-to 17-nucleotide "Tiny" RNAs

    Plant Cell

    (2019)
  • P.A. Bariola et al.

    The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation

    Plant J.

    (1994)
  • R.C. Edgar

    MUSCLE: multiple sequence alignment with high accuracy and high throughput

    Nucleic Acids Res.

    (2004)
  • Cited by (11)

    View all citing articles on Scopus
    View full text