The caleosin RD20/CLO3 regulates lateral root development in response to abscisic acid and regulates flowering time in conjunction with the caleosin CLO7.

The caleosins are encoded by multi-gene families in Arabidopsis thaliana and other plant species. This work investigates the role of two family members, RD20/CLO3 and CLO7, in flowering transition and in root development in response to ABA treatment. Gene expression of the caleosin RD20/CLO3 is induced by ABA in the root tissues and RD20/CLO3 has a negative affect on the total number of lateral roots as well as the length of the lateral roots in response to ABA treatment. The rd20/clo3 mutant has more and longer lateral roots in response to ABA treatment compared to the wild-type, showing that RD20/CLO3 plays a role in the ABA signaling pathway affecting this trait. In contrast, the caleosin CLO7 is not expressed in the roots and does not affect root architecture in response to ABA treatment. The disruption of both RD20/CLO3 and CLO7 together causes a dramatic early-flowering phenotype under long-day conditions, whereas single mutations in these genes do not affect flowering time under these conditions. Both yeast two-hybrid and bimolecular fluorescence complementation showed that both RD20/CLO3 and CLO7 interact with each other and can form homodimers and heterodimers. Taken together, these findings suggest that members of the caleosin gene family play both different and redundant roles in plant development.

The caleosin CLO7 and its role in the heterotrimeric G-protein signalling network.

The investigation of the caleosin CLO7 in relation to heterotrimeric G-protein signalling in Arabidopsis showed that the gene plays a role in seed germination and embryo viability. The caleosin CLO7 belongs to a multi-gene family of calcium-binding proteins which are characterized by single EF-hand motifs. Other members of the caleosin gene family have been shown to affect transpiration and seed germination as well as play a role in both abiotic and biotic stress responses. The proteins are associated with lipid droplets/oil bodies and some members of the gene family have been shown to have peroxygenase activity. Members of the gene family have also been shown to interact with the α subunit of the heterotrimeric G protein complex. In this study, we further expand on the diversity of physiological responses in which members of this gene family play regulatory roles. Utilizing BiFC and Y2H protein-protein interaction assays, CLO7 is identified as an interactor of the heterotrimeric G protein α subunit, GPA1. The full-length CLO7 is shown to interact with both the wild-type GPA1 and its constitutively active form, GPA1QL, at the plasma membrane. Point mutations to critical amino acids for calcium binding in the EF-hand of CLO7 indicate that the interaction with GPA1 is calcium-dependent and that the interaction with GPA1QL is enhanced by calcium. Protein-protein interaction assays also show that CLO7 interacts with Pirin1, a member of the cupin gene superfamily and a known downstream effector of GPA1, and this interaction is calcium-dependent. The N-terminal portion of CLO7 is responsible for these interactions. GFP-tagged CLO7 protein localizes to the endoplasmic reticulum (ER) and to lipid bodies. Characterization of the clo7 mutant line has shown that CLO7 is implicated in the abscisic acid (ABA) and mannitol-mediated inhibition of seed germination, with the clo7 mutant displaying higher germination rates in response to osmotic stress and ABA hormone treatment. These results provide insight into the role of CLO7 in seed germination in response to abiotic stress as well as its interaction with GPA1 and Pirin1. CLO7 also plays a role in embryo viability with the clo7gpa1 double mutant displaying embryo lethality, and therefore the double mutant cannot be recovered.

Characterization and expression of the Pirin gene family in Triticum aestivum.

Pirins are nuclear bicupin proteins, encoded by genes that are one of several gene families that comprise the cupin superfamily in plants. Pirin genes have been implicated in stress response pathways studied in Arabidopsis and At-Pirin1 has been shown to interact with the heterotrimeric G-protein alpha subunit (GPA1). The aim of this study was to identify the members of the Pirin gene family in Triticum aestivum, to correct their annotations in the whole genome, and gain an insight into their tissue-specific expression as well as their response to abiotic and biotic stresses. The Pirin gene family in T. aestivum is comprised of 18 genes that represent six paralogous gene copies, each having an A, B, and D homeolog. Expression analysis of the Pirin genes in T. aestivum Illumina RNA-seq libraries, which included sampling from differing tissue types as well as abiotic and biotic stresses, indicates that the members of the Pirin gene family have specialized expression and play a role in stress responses. Pirin gene families are also identified in other monocots including Aegilops tauschii, Hordeum vulgare, Brachypodium distachyon, Oryza sativa, Zea mays, Sorghum bicolor, and the dicot Arabidopsis thaliana.

Characterization of the heterotrimeric G protein gene families in Triticum aestivum and related species.

This study characterizes the heterotrimeric G protein gene families in Triticum aestivum, their tissue-specific expression patterns during development and in response to biotic and abiotic stress conditions. There are three Gα genes, three Gß and 12 Gγ genes, totaling 18 genes encoding heterotrimeric G proteins in the hexaploid wheat genome. Each haploid genome of the hexaploid T. aestivum has a single gene encoding the α subunit of the heterotrimeric G protein complex, GA1, a single Gß and four Gγ genes. Each gene has three homeologous copies in the A, B and D genomes. The physical interaction between the Gß (Gpb) and two Gγ subunits, Gpg1 and Gpg2, was shown through bimolecular fluorescence complementation (BiFC). The gene expression in response to biotic and abiotic stresses showed both up-regulation and down-regulation of members of the gene families. Gγ2-B and Gγ2-D are significantly upregulated during heat stress, GA1-D is upregulated by cold stress and Gγ1-A and Gγ1-D were upregulated by Fusarium graminearum inoculation in a F. graminearum resistant cultivar. This suggests that these members may play roles in biotic and abiotic signaling pathways and the roles of these genes within these pathways need further investigation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03156-9.

The stress induced caleosin, RD20/CLO3, acts as a negative regulator of GPA1 in Arabidopsis.

KEY MESSAGE: A stress induced calcium-binding protein, RD20/CLO3 interacts with the alpha subunit of the heterotrimeric G-protein complex in Arabidopsis and affects etiolation and leaf morphology. Heterotrimeric G proteins and calcium signaling have both been shown to play a role in the response to environmental abiotic stress in plants; however, the interaction between calcium-binding proteins and G-protein signaling molecules remains elusive. We investigated the interaction between the alpha subunit of the heterotrimeric G-protein complex, GPA1, of Arabidopsis thaliana with the calcium-binding protein, the caleosin RD20/CLO3, a gene strongly induced by drought, salt and abscisic acid. The proteins were found to interact in vivo by bimolecular fluorescent complementation (BiFC); the interaction was localized to the endoplasmic reticulum and to oil bodies within the cell. The constitutively GTP-bound GPA1 (GPA1QL) also interacts with RD20/CLO3 as well as its EF-hand mutant variations and these interactions are localized to the plasma membrane. The N-terminal portion of RD20/CLO3 was found to be responsible for the interaction with GPA1 and GPA1QL using both BiFC and yeast two-hybrid assays. RD20/CLO3 contains a single calcium-binding EF-hand in the N-terminal portion of the protein; disruption of the calcium-binding capacity of the protein obliterates interaction with GPA1 in in vivo assays and decreases the interaction between the caleosin and the constitutively active GPA1QL. Analysis of rd20/clo3 mutants shows that RD20/CLO3 plays a key role in the signaling pathway controlling hypocotyl length in dark grown seedlings and in leaf morphology. Our findings indicate a novel role for RD20/CLO3 as a negative regulator of GPA1.

Duplicated antagonistic EPF peptides optimize grass stomatal initiation.

Peptide signaling has emerged as a key component of plant growth and development, including stomatal patterning, which is crucial for plant productivity and survival. Although exciting progress has been made in understanding EPIDERMAL PATTERNING FACTOR (EPF) signaling in Arabidopsis, the mechanisms by which EPF peptides control different stomatal patterns and morphologies in grasses are poorly understood. Here, by examining expression patterns, overexpression transgenics and cross-species complementation, the antagonistic stomatal ligands orthologous to Arabidopsis AtEPF2 and AtSTOMAGEN/AtEPFL9 peptides were identified in Triticum aestivum (wheat) and the grass model organism Brachypodium distachyon. Application of bioactive BdEPF2 peptides inhibited stomatal initiation, but not the progression or differentiation of stomatal precursors in Brachypodium. Additionally, the inhibitory roles of these EPF peptides during grass stomatal development were suppressed by the contrasting positive action of the BdSTOMAGEN peptide in a dose-dependent manner. These results not only demonstrate how conserved EPF peptides that control different stomatal patterns exist in nature, but also suggest new strategies to improve crop yield through the use of plant-derived antagonistic peptides that optimize stomatal density on the plant epidermis.

Characterization of the Esi3/RCI2/PMP3 gene family in the Triticeae.

BACKGROUND: Members of the Early Salt Induced 3 (Esi3/RCI2/PMP3) gene family in plants have been shown to be induced in response to both biotic and abiotic stresses and to enhance stress tolerance in both transgenic plants and Saccharomyces cerevisiae. Esi3 was first identified as a salt stress induced gene in the salt tolerant wild wheat grass, Lophopyrum elongatum, and subsequently homologous genes in many other species were found to be members of the gene family. These include Arabidopsis thaliana and Oryza sativa where they are referred to as Rare Cold Inducible 2 (RCI2), and Zea mays where they are referred to as Plasma Membrane Protein 3 (PMP3). This study characterizes the Esi3 family members in Triticum aestivum and explores the tissue specific expression patterns of the gene family members as well as their response to a variety of environmental stresses. RESULTS: The Esi3 gene family was found to have a total of 29 family members comprised of ten paralogous groups in the hexaploid T. aestivum. Each paralogous group contains three homeologous copies, one in each of the A, B and D genomes with the exception of Esi3-2 which is missing the B copy. The genes of the Esi3 gene family were also identified in four other monocot species, Aegilops tauschii, Hordeum vulgare, Secale cereale and Sorghum bicolor, and were confirmed or corrected for Brachypodium distachyon, Oryza sativa and Zea mays, as well as the dicot Arabidopsis thaliana. Gene expression of the Esi3s was analyzed using tissue-specific, abiotic and biotic stress RNA-Seq 454 sequence libraries and Affymetrix microarray data for T. aestivum. CONCLUSIONS: Members of nearly all paralogous groups of the Esi3 genes in T. aestivum have altered gene expression in response to abiotic or biotic stress conditions. In addition, there are modest differences in gene expression among homeologous members of the gene family. This suggests that the Esi3 gene family plays an important role in the plants response to the stresses presented in this study. The Esi3-9 in T. aestivum has a unique N terminal extension placing it into Group III, a new group for the Esi3/RCI2/PMP3 gene family.

Characterization of the caleosin gene family in the Triticeae.

BACKGROUND: The caleosin genes encode proteins with a single conserved EF hand calcium-binding domain and comprise small gene families found in a wide range of plant species. Some members of the gene family have been shown to be upregulated by environmental stresses including low water availability and high salinity. Caleosin 3 from wheat has been shown to interact with the α-subunit of the heterotrimeric G proteins, and to act as a GTPase activating protein (GAP). This study characterizes the size and diversity of the gene family in wheat and related species and characterizes the differential tissue-specific expression of members of the gene family. RESULTS: A total of 34 gene family members that belong to eleven paralogous groups of caleosins were identified in the hexaploid bread wheat, T. aestivum. Each group was represented by three homeologous copies of the gene located on corresponding homeologous chromosomes, except the caleosin 10, which has four gene copies. Ten gene family members were identified in diploid barley, Hordeum vulgare, and in rye, Secale cereale, seven in Brachypodium distachyon, and six in rice, Oryza sativa. The analysis of gene expression was assayed in triticale and rye by RNA-Seq analysis of 454 sequence sets and members of the gene family were found to have diverse patterns of gene expression in the different tissues that were sampled in rye and in triticale, the hybrid hexaploid species derived from wheat and rye. Expression of the gene family in wheat and barley was also previously determined by microarray analysis, and changes in expression during development and in response to environmental stresses are presented. CONCLUSIONS: The caleosin gene family had a greater degree of expansion in the Triticeae than in the other monocot species, Brachypodium and rice. The prior implication of one member of the gene family in the stress response and heterotrimeric G protein signaling, points to the potential importance of the caleosin gene family. The complexity of the family and differential expression in various tissues and under conditions of abiotic stress suggests the possibility that caleosin family members may play diverse roles in signaling and development that warrants further investigation.