Unveiling the Secrets of Calcium-Dependent Proteins in Plant Growth-Promoting Rhizobacteria: An Abundance of Discoveries Awaits.

The role of Calcium ions (Ca2+) is extensively documented and comprehensively understood in eukaryotic organisms. Nevertheless, emerging insights, primarily derived from studies on human pathogenic bacteria, suggest that this ion also plays a pivotal role in prokaryotes. In this review, our primary focus will be on unraveling the intricate Ca2+ toolkit within prokaryotic organisms, with particular emphasis on its implications for plant growth-promoting rhizobacteria (PGPR). We undertook an in silico exploration to pinpoint and identify some of the proteins described in the existing literature, including prokaryotic Ca2+ channels, pumps, and exchangers that are responsible for regulating intracellular Calcium concentration ([Ca2+]i), along with the Calcium-binding proteins (CaBPs) that play a pivotal role in sensing and transducing this essential cation. These investigations were conducted in four distinct PGPR strains: Pseudomonas chlororaphis subsp. aurantiaca SMMP3, P. donghuensis SVBP6, Pseudomonas sp. BP01, and Methylobacterium sp. 2A, which have been isolated and characterized within our research laboratories. We also present preliminary experimental data to evaluate the influence of exogenous Ca2+ concentrations ([Ca2+]ex) on the growth dynamics of these strains.

Calcium-dependent protein kinase 2 plays a positive role in the salt stress response in potato.

KEY MESSAGE: StCDPK2 is an early player in the salt stress response in potato plants; its overexpression promoted ROS scavenging, chlorophyll stability, and the induction of stress-responsive genes conferring tolerance to salinity. The salinity of soils affects plant development and is responsible for great losses in crop yields. Calcium-dependent protein kinases (CDPKs) are sensor-transducers that decode Ca2+ signatures triggered by abiotic stimuli and translate them into physiological responses. Histochemical analyses of potato plants harboring StCDPK2 promoter fused to the reporter gene ß-glucuronidase (ProStCDPK2:GUS) revealed that GUS activity was high in the leaf blade and veins, it was restricted to root tips and lateral root primordia, and was observed upon stolon swelling. Comparison with ProStCDPK1:GUS and ProStCDPK3:GUS plants revealed their differential activities in the plant tissues. ProStCDPK2:GUS plants exposed to high salt presented enhanced GUS activity in roots which correlated with the numerous stress-responsive sites predicted in its promoter sequence. Moreover, StCDPK2 expression increased in in vitro potato plants after 2 h of high salt exposure and in greenhouse plants exposed to a dynamic stress condition. As inferred from biometric data and chlorophyll content, plants that overexpress StCDPK2 were more tolerant than wild-type plants when exposed to high salt. Overexpressing plants have a more efficient antioxidant system; they showed reduced accumulation of peroxide and higher catalase activity under salt conditions, and enhanced expression of WRKY6 and ERF5 transcription factors under control conditions. Our results indicate that StCDPK2 is an early player in the salt stress response and support a positive correlation between StCDPK2 overexpression and tolerance towards salt stress.

Characterization of two group III potato CDPKs, StCDPK22 and StCDPK24, that contain three EF-Hand motifs in their CLDs.

Four members of the potato (Solanum tuberosum L.) calcium-dependent protein kinase (CDPK) family StCDPK22/23/24 and StCDPK27, present three functional EF-hands motifs in their calmodulin-like domain (CLD). StCDPK22/23/24 are clustered in clade III-b1 with tomato and Arabidopsis CDPKs that lack the first EF-hand motif, while StCDPK27 is clustered in clade III-b3 with CDPKs that lack EF-hand 2. Members of each clade share similar intron-exon structures and acylation profiles. 3D model predictions suggested that StCDPK22 and StCDPK24 are active kinases that undergo a conformational switch in the presence of Ca2+ even when lacking one functional EF-hand motif; however, assays performed with recombinant proteins indicated that StCDPK24:6xHis was active in all the conditions tested, and its activity was enhanced in the presence of Ca2+, but StCDPK22:6xHis had scarce or null activity. Both kinases share with AtCPK8 the same autophosphorylation pattern in the autoinhibitory (AD) and C-terminal variable (CTV) domains, suggesting that it could be a characteristic of clade III-b1. RT-qPCR analysis revealed that StCDPK22 is mainly expressed in early stages of tuberization, but not limited to, while StCDPK24 expression is more ubiquitous. In silico analysis predicted several abiotic stress-responsive elements in its promoters. Accordingly, StCDPK24 expression peaked at 10 h in in vitro plants exposed to salt shock and then declined. Moreover, a significant increase was observed at 2 h in stems of salt-treated greenhouse plants, suggesting that this CDPK could participate in the early events of the signaling cascade triggered in response to salt.

Methylobacterium sp. 2A Is a Plant Growth-Promoting Rhizobacteria That Has the Potential to Improve Potato Crop Yield Under Adverse Conditions.

A Gram-negative pink-pigmented bacillus (named 2A) was isolated from Solanum tuberosum L. cv. Desirée plants that were strikingly more developed, presented increased root hair density, and higher biomass than other potato lines of the same age. The 16S ribosomal DNA sequence, used for comparative gene sequence analysis, indicated that strain 2A belongs to the genus Methylobacterium. Nucleotide identity between Methylobacterium sp. 2A sequenced genome and the rest of the species that belong to the genus suggested that this species has not been described so far. In vitro, potato plants inoculated with Methylobacterium sp. 2A had a better performance when grown under 50 mM NaCl or when infected with Phytophthora infestans. We inoculated Methylobacterium sp. 2A in Arabidopsis thaliana roots and exposed these plants to salt stress (75 mM NaCl). Methylobacterium sp. 2A-inoculated plants, grown in control or salt stress conditions, displayed a higher density of lateral roots (p < 0.05) compared to noninoculated plants. Moreover, under salt stress, they presented a higher number of leaves and larger rosette diameter. In dual confrontation assays, Methylobacterium sp. 2A displayed biocontrol activity against P. infestans, Botrytis cinerea, and Fusarium graminearum, but not against Rhizoctonia solani, and Pythium dissotocum. In addition, we observed that Methylobacterium sp. 2A diminished the size of necrotic lesions and reduced chlorosis when greenhouse potato plants were infected with P. infestans. Methylobacterium sp. 2A produces indole acetic acid, solubilizes mineral phosphate and is able to grow in a N2 free medium. Whole-genome sequencing revealed metabolic pathways associated with its plant growth promoter capacity. Our results suggest that Methylobacterium sp. 2A is a plant growth-promoting rhizobacteria (PGPR) that can alleviate salt stress, and restricts P. infestans infection in potato plants, emerging as a potential strategy to improve crop management.

StCDPK3 Phosphorylates In Vitro Two Transcription Factors Involved in GA and ABA Signaling in Potato: StRSG1 and StABF1.

Calcium-dependent protein kinases, CDPKs, decode calcium (Ca2+) transients and initiate downstream responses in plants. In order to understand how CDPKs affect plant physiology, their specific target proteins must be identified. In tobacco, the bZIP transcription factor Repression of Shoot Growth (NtRSG) that modulates gibberellin (GA) content is a specific target of NtCDPK1. StCDPK3 from potato is homologous (88% identical) to NtCDPK1 even in its N-terminal variable domain. In this work, we observe that NtRSG is also phosphorylated by StCDPK3. The potato RSG family of transcription factors is composed of three members that share similar features. The closest homologue to NtRSG, which was named StRSG1, was amplified and sequenced. qRT-PCR data indicate that StRSG1 is mainly expressed in petioles, stems, lateral buds, and roots. In addition, GA treatment affected StRSG1 expression. StCDPK3 transcripts were detected in leaves, petioles, stolons, roots, and dormant tubers, and transcript levels were modified in response to GA. The recombinant StRSG1-GST protein was produced and tested as a substrate for StCDPK3 and StCDPK1. 6xHisStCDPK3 was able to phosphorylate the potato StRSG1 in a Ca2+-dependent way, while 6xHisStCDPK1 could not. StCDPK3 also interacts and phosphorylates the transcription factor StABF1 (ABRE binding factor 1) involved in ABA signaling, as shown by EMSA and phosphorylation assays. StABF1 transcripts were mainly detected in roots, stems, and stolons. Our data suggest that StCDPK3 could be involved in the cross-talk between ABA and GA signaling at the onset of tuber development.

Characterization of StABF1, a stress-responsive bZIP transcription factor from Solanum tuberosum L. that is phosphorylated by StCDPK2 in vitro.

ABF/AREB bZIP transcription factors mediate plant abiotic stress responses by regulating the expression of stress-related genes. These proteins bind to the abscisic acid (ABA)-responsive element (ABRE), which is the major cis-acting regulatory sequence in ABA-dependent gene expression. In an effort to understand the molecular mechanisms of abiotic stress resistance in cultivated potato (Solanum tuberosum L.), we have cloned and characterized an ABF/AREB-like transcription factor from potato, named StABF1. The predicted protein shares 45-57% identity with A. thaliana ABFs proteins and 96% identity with the S. lycopersicum SlAREB1 and presents all of the distinctive features of ABF/AREB transcription factors. Furthermore, StABF1 is able to bind to the ABRE in vitro. StABF1 gene is induced in response to ABA, drought, salt stress and cold, suggesting that it might be a key regulator of ABA-dependent stress signaling pathways in cultivated potato. StABF1 is phosphorylated in response to ABA and salt stress in a calcium-dependent manner, and we have identified a potato CDPK isoform (StCDPK2) that phosphorylates StABF1 in vitro. Interestingly, StABF1 expression is increased during tuber development and by tuber-inducing conditions (high sucrose/nitrogen ratio) in leaves. We also found that StABF1 calcium-dependent phosphorylation is stimulated by tuber-inducing conditions and inhibited by gibberellic acid, which inhibits tuberization.

StCDPK2 expression and activity reveal a highly responsive potato calcium-dependent protein kinase involved in light signalling.

Calcium-dependent protein kinases (CDPKs) are essential calcium sensors. In this work, we have studied StCDPK2 isoform from potato both at gene and protein level. StCdpk2 genomic sequence contains eight exons and seven introns, as was observed for StCdpk1. There is one copy of the gene per genome located in chromosome 7. StCDPK2 encodes an active CDPK of 515 aminoacids, with an apparent MW of 57 kDa, which presents myristoylation and palmitoylation consensus in its N-terminus. StCDPK2 is highly expressed in leaves and green sprouts; enhanced expression was detected under light treatment, which corresponds well with light responsive cis-acting elements found in its promoter sequence. Antibodies against the recombinant StCDPK2::6xHis protein detected this isoform in soluble and particulate fractions from leaves. StCDPK2 autophosphorylation and kinase activity are both calcium dependent reaching half maximal activation at 0.6 µM calcium. The active kinase is autophosphorylated on serine and tyrosine residues and its activity is negatively modulated by phosphatidic acid (PA). Our results reveal StCDPK2 as a signalling element involved in plant growth and development and show that its activity is tightly regulated.

Genomic and functional characterization of StCDPK1.

StCDPK1 is a calcium dependent protein kinase expressed in tuberizing potato stolons and in sprouting tubers. StCDPK1 genomic sequence contains eight exons and seven introns, the gene structure is similar to Arabidopsis, rice and wheat CDPKs belonging to subgroup IIa. There is one copy of the gene per genome and it is located in the distal portion of chromosome 12. Western blot and immunolocalization assays (using confocal and transmission electron microscopy) performed with a specific antibody against StCDPK1 indicate that this kinase is mainly located in the plasma membrane of swelling stolons and sprouting tubers. Sucrose (4-8%) increased StCDPK1 protein content in non-induced stolons, however the amount detected in swelling stolons was higher. Transgenic lines with reduced expression of StCDPK1 (beta 7) did not differ from controls when cultured under multiplication conditions, but when grown under tuber inducing conditions some significant differences were observed: the beta 7 line tuberized earlier than controls without the addition of CCC (GA inhibitor), developed more tubers than wild type plants in the presence of hormones that promote tuberization in potato (ABA and BAP) and was more insensitive to GA action (stolons were significantly shorter than those of control plants). StCDPK1 expression was induced by GA, ABA and BAP. Our results suggest that StCDPK1 plays a role in GA-signalling and that this kinase could be a converging point for the inhibitory and promoting signals that influence the onset of potato tuberization.

Regulation of CDPK isoforms during tuber development.

CDPK activities present during tuber development were analysed. A high CDPK activity was detected in the soluble fraction of early stolons and a lower one was detected in soluble and particulate fractions of induced stolons. The early and late CDPK activities displayed diverse specificity for in vitro substrates and different subcellular distribution. Western blot analysis revealed two CDPKs of 55 and 60 kDa that follow a precise spatial and temporal profile of expression. The 55 kDa protein was only detected in early-elongating stolons and the 60 kDa one was induced upon stolon swelling, correlating with early and late CDPK activities. A new member of the potato CDPK family, StCDPK3, was identified from a stolon cDNA library. Gene specific RT-PCR demonstrated that this gene is only expressed in early stolons, while the previously identified StCDPK1 is expressed upon stolon swelling. This expression profile suggests that StCDPK3 could correspond to the 55 kDa isoform while StCDPK1 could encode the 60 kDa isoform present in swelling stolons. StCDPK1 has myristoylation and palmitoylation consensus possibly involved in its dual intracellular localization. Transient expression studies with wild-type and mutated forms of StCDPK1 fused to GFP were used to show that subcellular localization of this isoform is controlled by myristoylation and palmitoylation. Altogether, our data suggest that sequential activation of StCDPK3 and StCDPK1 and the subcellular localisation of StCDPK1 might be critical regulatory steps of calcium signalling during potato tuber development.

A calcium-dependent protein kinase is systemically induced upon wounding in tomato plants.

A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks.