Within and beyond the stringent response-RSH and (p)ppGpp in plants

Abstract

Plant RSH proteins are able to synthetize and/or hydrolyze unusual nucleotides called (p)ppGpp or alarmones. These molecules regulate nuclear and chloroplast transcription, chloroplast translation and plant development and stress response. Homologs of bacterial RelA/SpoT proteins, designated RSH, and products of their activity, (p)ppGpp-guanosine tetra-and pentaphosphates, have been found in algae and higher plants. (p)ppGpp were first identified in bacteria as the effectors of the stringent response, a mechanism that orchestrates pleiotropic adaptations to nutritional deprivation and various stress conditions. (p)ppGpp accumulation in bacteria decreases transcription-with exception to genes that help to withstand or overcome current stressful situations, which are upregulated-and translation as well as DNA replication and eventually reduces metabolism and growth but promotes adaptive responses. In plants, RSH are nuclei-encoded and function in chloroplasts, where alarmones are produced and decrease transcription, translation, hormone, lipid and metabolites accumulation and affect photosynthetic efficiency and eventually plant growth and development. During senescence, alarmones coordinate nutrient remobilization and relocation from vegetative tissues into seeds. Despite the high conservancy of RSH protein domains among bacteria and plants as well as the bacterial origin of plant chloroplasts, in plants, unlike in bacteria, (p)ppGpp promote chloroplast DNA replication and division. Next, (p)ppGpp may also perform their functions in cytoplasm, where they would promote plant growth inhibition. Furthermore, (p)ppGpp accumulation also affects nuclear gene expression, i.a., decreases the level of Arabidopsis defense gene transcripts, and promotes plants susceptibility towards Turnip mosaic virus. In this review, we summarize recent findings that show the importance of RSH and (p)ppGpp in plant growth and development, and open an area of research aiming to understand the function of plant RSH in response to stress.

Keywords: Alarmones; Photosynthesis; Plant growth and development; RelA/SpoT homologs; Senescence; Stress response.

Fig. 1

Schematic representation of the conserved domains structure in Arabidopsis thaliana RSH proteins. cTP, chloroplast target peptide (gray); HD, (p)ppGpp hydrolase domain (blue); SYNTH, (p)ppGpp synthetase domain; TGS, TGS regulatory domain; ACT (RRM), ACT regulatory domain (RNA recognition motif domain; orange); EFh, calcium binding EF hand motifs constituting on EF hand domain (yellow); synth G₃₇₆S, glycine into serine substitution at position 376 AA in the SYNTH domain that abolishes (p)ppGpp synthetase activity; HDc, degraded HD domain in CRSH, which may be nonfunctional (Atkinson et al. 2011); TM, putative transmembrane region (811–827 and 848–864 AA; black) and RPP5-ID, RPP5 interacting domain (634–793 AA) proposed by van der Biezen et al. (2000). The figure does not include the helical domain, which was found between TGS and ACT (RRM) domains, of RSH1 (Atkinson et al. 2011). Conserved domains localization is described based on the NCBI Conserved Domains database, except TGS domain in RSH2 and RSH3 proteins, which was found by Atkinson et al. (2011). According to Ito et al. (2017), ACT (RRM) (asterisk) and TGS (double asterisk) domains are not conserved in Arabidopsis RSH1 and RSH2/RSH3, respectively

Fig. 2

Electronic fluorescent pictographs (eFPs) for Arabidopsis thaliana RelA/SpoT homolog genes RSH1 (At4g02260), RSH2 (At3g14050), RSH3 (At1g54130) and RSH4 (At1g30850) transcript levels in different plant organs over various developmental stages. Expression levels between RSH are not normalized. High and no expression levels are indicated by red and yellow colors, respectively. For later stage siliques (6–10; corresponding to torpedo, walking-stick, curled and green cotyledons stages of embryo development) only the seeds were collected for analysis—not the siliques themselves. For stages 3–5 (corresponding to globular, heart and torpedo stages of embryo development) the seeds were collected with siliques. More detailed information about the microarray and other studies that are the sources for these developmental maps and further tissue-related information can be found at Arabidopsis eFP Browser at bar.utoronto.ca, in Gene Expression Map of Arabidopsis development (Schmid et al. and the Nambara Lab) and Winter et al. (2007)

Fig. 3

Electronic fluorescent pictographs (eFPs) for Arabidopsis thaliana RSH1 (At4g02260), RSH2 (At3g14050), RSH3 (At1g54130) and RSH4 (At1g30850) transcript levels under different stresses. 18 days after sowing and 3 h after dark/light transition Arabidopsis Col-0 plants treated with cold (plants transferred to ice for rapid cooling and kept at 4 °C in the cold room until harvest), 300 mM mannitol (osmotic stress), 150 mM NaCl (salt stress), drought (plants exposed to air stream for 15 min with loss of approximately 10% fresh weight), 10 μM methyl viologen (oxidative stress), wounding (leaves punctuation) or heat (38 °C for 3 h followed by recovery at 25 °C until harvest) conditions were harvested at 1 and 12 h post treatment. Expression levels between RSH are not normalized. High and no expression levels are indicated by red and yellow colors, respectively. More detailed information about the microarray and other studies that are the sources for these stress response maps and further information can be found at Arabidopsis eFP Browser at bar.utoronto.ca, in Winter et al. (2007) and Kilian et al. (2007)

Fig. 4

Overview of a proposed model for the plant stringent response. RSH1–CRSH are nuclear genes, whose expressions change during plant development and under stress. Based on RSH gene and protein expression studies as well as (p)ppGpp accumulation data under stress or hormone application, we propose that the stringent response in plants can be modulated by pathogens, wounding, UV irradiation, heat shock, cold stress, salinity, drought, exogenous hormone application (JA, jasmonic acid; ET, ethylene; SA, salicylic acid; ABA, abscisic acid: AUX, auxin), heavy metals, osmotic, oxidative stress and is additionally regulated by diurnal rhythms. Stress or hormone names written in italics were shown to increase (p)ppGpp concentration in plants. In that particular study (Takahashi et al. 2004), only the application of cold did not affect the level of alarmones, whereas SA, pathogens and osmotic and oxidative stress were not checked for the induction of (p)ppGpp production. Interestingly, exogenous auxin application blocks (p)ppGpp accumulation in pea plants (purple color). Since SA, JA and ET mediate pathogen-invoked responses, JA and ET wounding and heat shock-invoked responses, ET and ABA heat shock-prompted responses and ABA salt and drought-invoked events, these hormones were proposed to mediate the stress-induced plant stringent response, likely via regulation of the RSH gene expression. RSH proteins are translocated into chloroplasts, as they carry the chloroplastic transit peptide (cTP), where they regulate (p)ppGpp metabolism. RSH2/RSH3 along with CRSH function as the major (p)ppGpp synthetases and produce ppGpp and pppGpp from GDP and GTP, respectively, whereas RSH1 appears to be the main (p)ppGpp hydrolase. Possibly, (p)ppGpp also accumulate in cytoplasm either being produced there, before RSH translocation to chloroplasts, or in chloroplasts, from where they are transported to cytoplasm. Recently, plant genes encoding for possible pppGpp-specific phosphatases GppA/Ppx, which may function in cytoplasm to convert pppGpp to ppGpp, had been reported. In chloroplasts, (p)ppGpp affects transcription, downregulating the expression of PEP- and probably also NEP-dependent genes encoding for the elements of, e.g., PSI, PSII, translation machinery and others (table on the right bottom corner, blue color indicates decreased gene expression; functions of genes are described in gray fields). The regulation of transcription occurs either due to decrease in GTP level, a nucleotide important for the initiation of transcription from rRNA genes, for the sake of (p)ppGpp production, or via direct (p)ppGpp–PEP interaction. (p)ppGpp accumulation also influences nuclear gene expression (table on the left bottom corner, blue and red colors indicate decreased and increased gene expression, respectively). It promotes the expression of genes important for chloroplast functioning, cytosolic rRNA processing as well as JA-responsive. However, it leads to the reduction of transcripts of defense-related genes, such as LRR receptor kinases and MPK/MKK that serve to recognize microorganism associated molecular patterns and are important for signal transduction, respectively. It also downregulates the expression of genes encoding proteins involved in SA biosynthesis and signaling as well as responsive to the hormone. (p)ppGpp also regulate chloroplast translation, likely indirectly, as an effect of the negative regulation of transcription. However, (p)ppGpp may also inhibit translation directly, e.g., via binding to the elongation factor G. Chloroplast proteins found to be downregulated under (p)ppGpp accumulation are marked in the right bottom corner table in bold (PsaB, PsaA, RbcL). Additionally, the Rubisco small subunit (nucleus-encoded) and chloroplast f1 proteins (both NEP- and PEP-dependent), whose gene expression under (p)ppGpp accumulation was not shown so far, were assessed as downregulated under (p)ppGpp accumulation. Nuclear-encoded PR1 protein level was also shown as downregulated under (p)ppGpp over accumulation (marked in bold); however, it is likely the effect of decreased expression of the gene. (p)ppGpp inhibit GKs and ASs involved in GTP and ATP metabolism, respectively, and thus downregulate purine biosynthesis as well. Furthermore, (p)ppGpp accumulation in chloroplasts decreases SA level, the total lipids content as well as the levels of many other metabolites (purple color). In plants, many hormonal and environmental signals raise intercellular concentration of Ca²⁺, a messenger regulating cellular and developmental processes via Ca²⁺-binding proteins. One such protein is CRSH, whose (p)ppGpp synthetic activity depends on the calcium ion concentration. Since both cytosol and chloroplasts are loci of Ca²⁺ accumulation in plants, it is not clear whether CRSH is activated only via chloroplastic or both chloroplastic and cytosolic Ca²⁺ pools. PEP—plastid-encoded plastid RNA polymerase, NEP—nuclear-encoded plastid RNA polymerase, PSI—photosystem I, PSII—photosystem II, rps14—ribosomal protein 14, TRNR—arginine tRNA, rbcL—Rubisco large subunit, rpoA—RNA polymerase alpha subunit, rpoB—RNA polymerase beta subunit, clpP1—caseinolytic protease P1, ycf1—a subunit of the translocon on the inner envelope of chloroplasts, rps18—ribosomal protein 18, accD—acetyl-CoA carboxylase beta subunit, ycf2—ATPase of unknown function (UF), FRK1—Flg22-induced receptor-like kinase 1, CERK1—chitin elicitor receptor kinase 1, NIK2—NSP interacting kinase 2, SOBIR1—suppresor of BIR1-1, MPK (MAPK)—mitogen-activated protein kinase, MPKK—MPK kinase, ICS1—isochorismate synthase 1, CBP60g—calmodulin binding protein 60g, SARD1—SAR deficient 1, NPR1—non-expressor of PR genes 1, PR1—pathogenesis-related 1, PR2—pathogenesis-related 2, PR5—pathogenesis-related 5, AS—adenylosuccinate synthetase, GK—guanylate kinase