Immunolocalization and histochemical evidence for the association of two different<Emphasis Type="Italic"> Arabidopsis</Emphasis> annexins with secretion during early seedling growth and development

Annexins are a multigene, multifunctional family of calcium-dependent, membrane-binding proteins found in animal and plant cells. In plants, annexins have been localized in the cytoplasm and at the cell periphery of highly secretory cell types, and in the tip region of polarly growing cells. Consequently, one proposed function for annexins in plant cells is participation in the Golgi-mediated secretion of new wall materials. In Arabidopsis, there are eight different annexin cDNAs, which share between 30% and 81% deduced amino acid sequence identity. We have used two monospecific Arabidopsis anti-annexin antibodies, raised against divergent 31-mer peptides from AnnAt1 and AnnAt2 and a previously characterized pea anti-annexin p35 antibody, for Western blot and immunolocalization studies in Arabidopsis. Western blot analyses of various Arabidopsis protein fractions showed that the two Arabidopsis antibodies are able to specifically recognize annexins in both soluble and membrane fractions. Immunofluorescence results with the three annexin antibodies show staining of secretory cells, especially at the cell periphery in developing sieve tubes, outer root cap cells, and in root hairs, consistent with previous results. In developmentally different stages some staining was also seen near the apical meristem, in some leaf cells, and in phloem-associated cells. Autoradiography following ³H-galactose incorporation was used to more clearly correlate active secretion of wall materials with the localization patterns of a specific individual annexin protein in the same cells at the same developmental stage. The results obtained in this study provide further support for the hypothesis that these two Arabidopsis annexins function in Golgi-mediated secretion during early seedling growth and development.     

Potential role of annexin AnnAt1 from Arabidopsis thaliana in pH-mediated cellular response to environmental stimuli

Plant annexins, Ca(2+)- and membrane-binding proteins, are probably implicated in the cellular response to stress resulting from acidification of cytosol. To understand how annexins can contribute to cellular ion homeostasis, we investigated the pH-induced changes in the structure and function of recombinant annexin AnnAt1 from Arabidopsis thaliana. The decrease of pH from 7.0 to 5.8 reduced the time of the formation of ion channels by AnnAt1 in artificial lipid membranes from 3.5 h to 15-20 min and increased their unitary conductance from 32 to 63 pS. These changes were accompanied by an increase in AnnAt1 hydrophobicity as revealed by hydrophobicity predictions, by an increase in fluorescence of 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS) bound to AnnAt1 and fluorescence resonance energy transfer from AnnAt1 tryptophan residues to TNS. Concomitant lipid partition of AnnAt1 at acidic pH resulted in its partial protection from proteolytic digestion. Secondary structures of AnnAt1 determined by circular dichroism and infrared spectroscopy were also affected by lowering the pH from 7.2 to 5.2. These changes were characterized by an increase in beta-sheet content at the expense of alpha-helical structures, and were accompanied by reversible formation of AnnAt1 oligomers as probed by ultracentrifugation in a sucrose gradient. A further decrease of pH from 5.2 to 4.5 or lower led to the formation of irreversible aggregates and loss of AnnAt1 ionic conductance. Our findings suggest that AnnAt1 can sense changes of the pH milieu over the pH range from 7 to 5 and respond by changes in ion channel conductance, hydrophobicity, secondary structure of the protein and formation of oligomers. Further acidification irreversibly inactivated AnnAt1. We suggest that the pH-sensitive ion channel activity of AnnAt1 may play a role in intracellular ion homeostasis.     

Two novel annexins from Drosophila melanogaster. Cloning, characterization, and differential expression in development

The annexins are a family of homologous Ca2(+)- and phospholipid-binding proteins that until now have only been found in vertebrates. cDNA clones encoding two novel annexins from Drosophila melanogaster were isolated and characterized. RNA blots indicate that the messages for the two Drosophila proteins are differentially expressed in development, with one message being expressed throughout development, while the other is only found in early embryos and adult flies. In situ hybridizations localize the two Drosophila genes to 93B and 19A-4,7. A similarly high degree of homology relates Drosophila annexins to different vertebrate annexins, indicating that the Drosophila annexins are not the invertebrate homologues of particular mammalian annexins but that they constitute novel members of the annexin gene family. In continuation with a recently established terminology, the Drosophila annexins will be named annexins IX and X. The biochemical properties of Drosophila annexin X were investigated using recombinant protein. Similar to vertebrate annexins, annexin X bound to liver membranes and liposomes containing phosphatidylserine in a calcium-dependent manner but not to liposomes containing phosphatidylcholine. In addition, annexin X partitioned into the detergent phase of Triton X-114 as a function of calcium. The conservation of the annexin family of Ca2(+)-binding proteins in invertebrates suggests that they have a basic function in cells which is not peculiar to vertebrate biology, and the availability of the Drosophila sequences will open avenues for mutational studies of these functions.     

Participation of annexin At1 in plant response to abiotic stress

Annexins are calcium-dependent phospholipid-binding proteins existing both in animal and plant cells. Mammalian and especially human annexins were examined for many years, and their functions in these organisms are already well known, but it is not the case for plant annexins. On the basis of existing literature and experimental evidence, it can be proposed that plant annexins may have a role in stress response. Annexin At1 of Arabidopsis thaliana (AnnAt1) is one of eight proteins of this family in A. thaliana. In its sequence many potentially functional domains are found, owing to that this protein can play an important role in stress response of the organism. Considering literature data and our own experiments one can postulate that AnnAt1 has weak peroxidase activity and form oligomers in hydrogen peroxide-dependent manner. This can be important in response to oxidative stress. Also we found that this protein forms ion channel in pH-dependent manner. This phenomenon may have particular significance in maintaining calcium homeostasis in the cell and calcium signaling, therefore AnnAt1 may play different roles in regulating stress response of plant. This is extremely important because plants during growth and development have to cope different stress factors like drought, deficiency or excess of mineral compounds in the soil, as well as low or high temperatures.     

Peroxidase activity of annexin 1 from Arabidopsis thaliana

On the basis of earlier reports suggesting that annexin A1 from Arabidopsis thaliana (AnnAt1) participates in limiting the excessive levels of reactive oxygen species during oxidative burst in plants, we examined the sensitivity of recombinant AnnAt1 to hydrogen peroxide and its peroxidase activity. Purified recombinant protein remains mostly alpha-helical and binds to lipids in a calcium-dependent manner. Upon oxidation recombinant AnnAt1 exhibits a tendency to form dimers in vitro. AnnAt1 is also sensitive to the presence of reducing agents, suggesting that AnnAt1 is a redox sensor in plant cells. Moreover, using two independent methods we found that AnnAt1 displayed peroxidase activity which is probably related to the presence of a heme-binding domain within AnnAt1, as present in other peroxidases. Indeed, site-directed mutagenesis within this domain resulted in a complete abrogation of the activity of AnnAt1. Furthermore, this activity was found to be sensitive to the phosphorylation state of the protein.     

Secondary product glucosyltransferase and putative glucosyltransferase expression during Citrus paradisi (c.v. Duncan) growth and development

Flavonoids are secondary metabolites that have significant roles in plant defense and human nutrition. Glucosyltransferases (GTs) catalyze the transfer of sugars from high energy sugar donors to other substrates. Several different secondary product GTs exist in the tissues of grapefruit making it a model plant for studying their structure and function. The goal of this investigation was to determine the expression patterns of seven putative secondary product GTs during grapefruit growth and development by quantifying mRNA expression levels in the roots, stems, leaves, flowers, and mature fruit to establish whether the genes are expressed constitutively or if one or more could be expressed in a tissue specific manner and/or developmentally regulated. Six growth stages were defined from which RNA was extracted, and expression levels were quantified by standardized densitometry of gene-specific RT-PCR products. Results show that there were variable degrees of PGT expression in different tissues and at different developmental stages. These results add to the growing knowledge base of dynamics of expression and potential regulation of secondary metabolism in Citrus paradisi.     

Characterization, physical location and expression of the genes encoding calcium/calmodulin-dependent protein kinases in maize (Zea mays L.)

The maize genomic sequence and cDNA encoding a calcium/calmodulin-dependent protein kinase homolog were isolated and identified. The deduced peptide (MCK2) from this cDNA shared high amino acid identity (91.2%) with maize MCK1. These two genes were physically mapped onto chromosomes by fluorescence in situ hybridization using the first introns of the genes as gene-specific probes. While the MCK1 gene was assigned to a locus on the long arm of chromosome 9, the MCK2 gene was localized to a locus on the long arm of chromosome 1. Both of these genes were expressed in roots, leaves, stems and flowers, and the expression patterns of MCK were verified by RNA in situ hybridization. These results indicated that MCK expression is temporally and spatially regulated during maize growth and development.     

Characterization of Annexin gene family and functional analysis of RsANN1a involved in heat tolerance in radish (Raphanus sativus L.)

Plant annexins are a kind of conserved Ca²⁺-dependent phospholipid-binding proteins which are involved in plant growth, development and stress tolerance. Radish is an economically important annual or biennial root vegetable crop worldwide. However, the genome-wide characterization of annexin (RsANN) gene family remain largely unexplored in radish. In this study, a comprehensive identification of annexin gene family was performed at the whole genome level in radish. In total, ten RsANN genes were identified, and these putative RsANN proteins shared typical characteristics of the annexin family proteins. Phylogenetic analysis showed that the RsANNs together with annexin from Arabidopsis and rice were clustered into five groups with shared similar motif patterns. Chromosomal localization showed that these ten RsANN genes were distributed on six chromosomes (R3-R8) of radish. Several cis-elements involved in abiotic stress response were identified in the promoter regions of RsANN genes. Expression profile analysis indicated that the RsANN genes exhibited tissue-specific patterns at different growth stages and tissues. The Real-time quantitative PCR (RT-qPCR) revealed that the expression of most RsANN genes was induced under various abiotic stresses including heat, drought, salinity, oxidization and ABA stress. In addition, stress assays showed that overexpression of RsANN1a improved plant’s growth and heat tolerance, while artificial microRNAs (amiRNA)-mediated knockdown of RsANN1a caused dramatically decreased survival ratio of Arabidopsis plants. These findings not only demonstrate that RsANN1a might play a critical role in the heat stress response of radish, but also facilitate clarifying the molecular mechanism of RsANN genes in regulating the biological process governing plant growth and development.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01056-5.     

Genomic organization, phylogenetic comparison and expression profiles of annexin gene family in tomato (Solanum lycopersicum)

Annexins have been suggested to play pivotal roles in stress resistance and plant development. However, related studies on fruit-bearing plants, especially on fruit development, are very limited. In the present study, we provide a comprehensive overview of the annexin family in tomato, describing the gene structure, promoter cis-regulatory elements, organ expression profile, and gene expression patterns under hormone and stress treatments. Bioinformatic analysis revealed that the nine tomato annexins were structurally different from their animal counterparts, but highly conserved annexin domains were still found in most of them. Cis-regulatory element prediction showed that there were important elements in the 2kb upstream promoter regions, including stress- and hormone-responsive-related elements. The expression patterns of these genes were investigated, and the results revealed that they were regulated under developmental processes and environmental stimuli. Among them, AnnSl1.1 and AnnSl2 were highly expressed in most of the tested organs. Genes preferentially or specifically expressed in organs, such as stigma or ovary (AnnSl6), stamen (AnnSl8), and fruit pericarp (AnnSl1.2 and AnnSl9), were identified. Some annexin genes were induced by plant hormones including abscisic acid (AnnSl3, AnnSl6, AnnSl8, and AnnSl9) and gibberellic acid (AnnSl1.1, AnnSl1.2, AnnSl4, and AnnSl7). Most of these annexin genes were induced by salt, drought, wounding, and heat or cold stresses. The present study provides significant information for understanding the diverse roles of annexins in tomato growth and development.     

Distinct Annexin Subfamilies in Plants and Protists Diverged Prior to Animal Annexins and from a Common Ancestor

Annexin homologues in the kingdoms of Planta and Protista were characterized by molecular sequence analysis to determine their phylogenetic and structural relationship with annexins of Animalia. Sequence fragments from 19 plant annexins were identified in sequence databases and composite sequences were also assembled from expressed sequence tags for Arabidopsis thaliana. Length differences in protein amino-termini and evidence for unique exon splice sites indicated that plant annexins were distinct from those of animals. A third annexin gene of Giardia lamblia (Anx21-Gla) was identified as a distant relative to other protist annexins and to those of higher eukaryotes, thus providing a suitable outgroup for evolutionary reconstruction of the family tree. Rooted evolutionary trees portrayed protist, plant, and Dictyostelium annexins as early, monophyletic ramifications prior to the appearance of closely related animal annexin XIII. Molecular phylogenetic analyses of DNA and protein sequence alignments revealed at least seven separate plant subfamilies, represented by Anx18 (alfalfa, previously classified), Anx22 (thale cress), Anx23 (thale cress, cotton, rape and cabbage), Anx24 (bell pepper and tomato p34), Anx25 (strawberry, horseradish, pea, soybean, and castor bean), Anx26-Zma, and Anx27-Zma (maize). Other unique subfamilies may exist for rice, tomato p35, apple, and celery annexins. Consensus sequences compiled for each eukaryotic kingdom showed some breakdown of the ``annexin-fold' motif in repeats 2 and 3 of protist and plant annexins and a conserved codon deletion in repeat 3 of plants. The characterization of distinct annexin genes in plants and protists reflects their comparable diversity among animal species and offers alternative models for the comparative study of structure–function relationships within this important gene family. Received: 30 May 1996 / Accepted: 20 August 1996     

Induction of annexin by heavy metals and jasmonic acid in Zea mays

Plant annexins are Ca²⁺- and phospholipid-binding proteins forming an evolutionary conserved multi-gene family. They are implicated in the regulation of plant growth, development, and stress responses. With the availability of the maize genome sequence information, we identified 12 members of the maize annexin genes. Analysis of protein sequence and gene structure of maize annexins led to their classification into five different orthologous groups. Expression analysis by RT-PCR revealed that these genes are responsive to heavy metals (Ni, Zn, and Cd). The maize annexin genes were also found to be regulated by Ustilago maydis and jasmonic acid. Additionally, the promoter of the maize annexin gene was analyzed for the presence of different stress-responsive cis-elements, such as ABRE, W-box, GCC-box, and G-box. RT-PCR and microarray data show that all 12 maize annexin genes present differential, organ-specific expression patterns in the maize developmental steps. These results indicate that maize annexin genes may play important roles in the adaptation of plants to various environmental stresses.