Callose Levels Research Articles

An efficient method for the extraction and the quantitative determination of callose from HLB-affected and healthy citrus

Callose is a 1,3-β glucan responsible for several processes in planta such as cell division, ripening of pollen mother cells, maintaining the plasmodesmata opening, and providing structure to the sieve plates. Besides the physiological roles, callose is deposited during pathogen attacks, forming papillae to prevent pathogen entrance into the tissue or plugging the sieve pores to limit the spread of phloem pathogens. Candidatus Liberibacter asiaticus (CLas), the putative causal agent of Huanglongbing (HLB), is a phloem-restricted pathogen whose infection leads to the production of callose in the phloem. Characterizing the dynamics of callose plugging during HLB is important for understanding the disease, but no published protocols are available for the extraction and quantification of the callose in citrus trees, and quantitative data are limited. Detection of callose by microscopy is time-consuming and expensive and does not give information about the distribution of callose in the entire plant. Here, we present a short protocol for the efficient extraction and quantification of the total callose from citrus plants. We compared different tissues from healthy and CLas-infected plants and identified an increase in callose levels in the midribs, the stems, and the fruit peduncles of infected plants. Callose levels were highest in leaves, especially midribs, compared to stems, roots, and fruit peduncles. This method could be applied to other woody plant species.

Stem-pitting caused by Citrus tristeza virus is associated with increased phloem occlusion

Stem-pitting (SP) disease results from disruption of normal phloem and xylem development. In citrus, a characteristic manifestation of SP caused by Citrus tristeza virus (CTV) is phloem regeneration. We hypothesized that phloem regeneration occurs due to reduced functionality of CTV infected phloem cells. To examine phloem cell occlusions in CTV-SP, we analyzed callose and phloem-protein (PP) accumulation in Citrus macrophylla trees infected with CTV mutants exhibiting different SP phenotypes from very mild (CTVΔp13) to severe (CTVΔp33), in addition to full-length CTV and healthy plants. CTV infection was accompanied by callose and PP accumulation in the phloem. With the increase in the SP symptoms from very mild to severe, there was a constant increase in the levels of callose and PP, accompanied by an increase in PHLOEM-PROTEIN 2 and a decrease in BETA-1,3-GLUCANASE gene expression levels. These results indicate that SP symptom development is associated with increased phloem occlusion.

Unraveling the transcriptional network regulated by miRNAs in blast-resistant and blast-susceptible rice genotypes during Magnaporthe oryzae interaction

The plant pathogen Magnaporthe oryzae poses a significant threat to global food security, and its management through the cultivation of resistant varieties and crop husbandry practices, including fungicidal sprays, has proven to be inadequate. To address this issue, we conducted small-RNA sequencing to identify the roles of miRNAs and their target genes in both resistant (PB1637) and susceptible (PB1) rice genotypes. We confirmed the expression of differentially expressed miRNAs using stem-loop qRT-PCR analysis and correlated them with rice patho-phenotypic and physio-biochemical responses. Our findings revealed several noteworthy differences between the resistant and susceptible genotypes. The resistant genotype exhibited reduced levels of total chlorophyll and carotenoids compared to the susceptible genotype. However, it showed increased levels of total protein, callose, H2O2, antioxidants, flavonoids, and total polyphenols. Additionally, among the defense-associated enzymes, guaiacol peroxidase and polyphenol oxidase responses were higher in the susceptible genotypes. In our comparative analysis, we identified 27 up-regulated and 43 down-regulated miRNAs in the resistant genotype, while the susceptible genotype exhibited 44 up-regulated and 62 down-regulated miRNAs. Furthermore, we discovered eight up-regulated and five down-regulated miRNAs shared between the resistant and susceptible genotypes. Notably, we also identified six novel miRNAs in the resistant genotype and eight novel miRNAs in the susceptible genotype. These novel miRNAs, namely Chr8_26996, Chr12_40110, and Chr12_41899, were found to negatively correlate with the expression of predicted target genes, including Cyt-P450 monooxygenase, serine carboxypeptidase, and zinc finger A20 domain-containing stress-associated protein, respectively. The results of our study on miRNA and transcriptional responses provide valuable insights for the development of future rice lines that are resistant to blast disease. By understanding the roles of specific miRNAs and their target genes in conferring resistance, we can enhance breeding strategies and improve crop management practices to ensure global food security.

Average daily temperature controls floral bud formation rate, callose deposition and flower development of Hydrangea macrophylla ‘Early Blue’

ABSTRACT Hydrangea macrophylla ‘Early Blue’ was exposed to six different day (DT) and night (NT) and average daily temperatures (ADT) during flower bud formation (DT/NT°C: 12/12, 17/12, 17/17, 22/12, 22/17, 22/22). The aim was to evaluate the effect of ADT and DT/NT on the rate of bud formation and forcing time after a period of chilling. Furthermore, the role of callose in dormancy induction and release during cold storage was investigated. ADT ≤ 17°C accelerated flower bud formation irrespective of DT and NT compared to an ADT > 17°C. Floral buds developed at ADT 17°C contained significantly lower level of callose compared to buds from the higher ADT regimes (ADT >17°C). After 6 weeks of chilling (10°C) the callose level was decreased to a similar level irrespective of the DT/NT regime during floral bud formation and/or floral stage at the start of the chilling treatment. We conclude that callose may play an important role in the dormancy release of H. macrophylla. However, floral buds do not have to be fully developed to remove the callose in response to chilling and no clear relationship was found between the levels of callose after chilling and forcing time.

Evaluation of host resistance and susceptibility to Podosphaera aphanis NWAU1 infection in 19 strawberry varieties

Strawberry powdery mildew, caused by the obligate biotrophic fungus Podosphaera aphanis, is one of the most devastating diseases in strawberry, resulting in severe reduction in fruit yield and quality. Exploring and utilizing powdery mildew-resistant strawberries is an important disease management strategy. The present study evaluated the resistance of 19 strawberry varieties to Podosphaera aphanis NWAU1 (Pa. NWAU1) based on disease index. We have comprehensively investigated the different cytological and physiological responses of different resistant strawberries. Compared with highly susceptible (HS) strawberries 'Benihoppe' and 'Tianxianzui', the moderately resistant (MR) strawberries 'Heilongjiang' and 'Yellow Wonder' exhibited higher levels of callose and H2O2 accumulation after inoculation with Pa. NWAU1. The activities of SOD, POD, GPX, and the GSH level in HS strawberries were increased after inoculation with Pa. NWAU1. The SA and JA levels in resistant strawberries were almost similar. However, the ACC level varied in HS and MR strawberries. Our results indicate that increased levels of callose, H2O2 and ACC resulted in resistance to Pa. NWAU1 in MR strawberries. The higher antioxidant enzyme activities may contribute to the sensitivity to Pa. NWAU1 in HS strawberries. Our study provides a reliable and comprehensive method for evaluating and understanding potential defense responses to powdery mildew resistance in different strawberries.

Xanthomonas campestris pv. musacearum Bacterial Infection Induces Organ-Specific Callose and Hydrogen Peroxide Production in Banana

Xanthomonas campestris pv . musacearum ( Xcm) bacteria cause banana Xanthomonas wilt (BXW), the most destructive disease of bananas in East and Central Africa. During early stages of infection in susceptible banana cultivars, incomplete systemic movement of Xcm limits bacterial colonization in the upper organs. The mechanistic basis of this delayed movement is unknown. We hypothesized that Xcm infection triggers basal pattern-triggered immune (PTI) responses whose spatial and temporal variability along the banana's anatomical structure accounts for initially limiting Xcm in upper organs. Hence, we examined PTI responses such as callose deposition and hydrogen peroxide (H2O2) production in different organs in response to Xcm infection in BXW-susceptible Kayinja and Mbwazirume banana cultivars and wild resistant progenitor Musa balbisiana. Xcm-induced callose increased and peaked at 14 days postinoculation (dpi) and 28 dpi as assessed by fluorescence microscopy and enzyme-linked immunosorbent assays, respectively. The levels of Xcm-induced H2O2 and callose were highest in the pseudostems and corms, respectively, and were independent of host susceptibility or resistance to BXW. H2O2 production showed a biphasic transient pattern with an initial increase at 1-hour post Xcm inoculation (hpi), followed by a decline 3 to 6 hpi and then a second increase by 12 hpi. Our findings point to organ-specific responses to Xcm infection in bananas. The corm, which doubles as a subterranean perennating organ and interface between mother plants and lateral shoots, was the most responsive organ in callose production, whereas the pseudostem was the most responsive organ in H2O2 production, suggesting the significance of these organs in banana response to BXW. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Sandwich Enzyme-Linked Immunosorbent Assay for Quantification of Callose.

The existing methods of callose quantification include epifluorescence microscopy and fluorescence spectrophotometry of aniline blue-stained callose particles, immuno-fluorescence microscopy and indirect assessment of both callose synthase and β-(1,3)-glucanase enzyme activities. Some of these methods are laborious, time consuming, not callose-specific, biased and require high technical skills. Here, we describe a method of callose quantification based on Sandwich Enzyme-Linked Immunosorbent Assay (S-ELISA). Tissue culture-derived banana plantlets were inoculated with Xanthomonas campestris pv. musacearum (Xcm) bacteria as a biotic stress factor inducing callose production. Banana leaf, pseudostem and corm tissue samples were collected at 14 days post-inoculation (dpi) for callose quantification. Callose levels were significantly different in banana tissues of Xcm-inoculated and control groups except in the pseudostems of both banana genotypes. The method described here could be applied for the quantification of callose in different plant species with satisfactory level of specificity to callose, and reproducibility. Additionally, the use of 96-well plate makes this method suitable for high throughput callose quantification studies with minimal sampling and analysis biases. We provide step-by-step detailed descriptions of the method.

In Vivo Aniline Blue Staining and Semiautomated Quantification of Callose Deposition at Plasmodesmata.

The deposition and turnover of callose (beta-1,3 glucan polymer) in the cell wall surrounding the neck regions of plasmodesmata (PD) controls the cell-to-cell diffusion rate of molecules and, therefore, plays an important role in the regulation of intercellular communication in plants.Here we describe a simple and fast in vivo staining procedure for the imaging and quantification of callose at PD. We also introduce calloseQuant, a plug-in for semiautomated image analysis and non-biased quantification of callose levels at PD using ImageJ.

Callose Detection and Quantification at Plasmodesmata in Bryophytes.

In bryophytes (i.e., mosses, liverworts, and hornworts), extant representatives of early land plants, plasmodesmata have been described in a wide range of tissues. Although their contribution to bryophyte morphogenesis remains largely unexplored, several recent studies have suggested that the deposition of callose around plasmodesmata might regulate developmental and physiological responses in mosses. In this chapter, we provide a protocol to image and quantify callose levels in the filamentous body of the model moss Physcomitrium (Physcomitrella) patens and discuss possible alternatives and pitfalls. More generally, this protocol establishes a framework to explore the distribution of callose in other bryophytes.

The Origin of the Cyathea delgadii Sternb. Somatic Embryos Is Determined by the Developmental State of Donor Tissue and Mutual Balance of Selected Metabolites.

Somatic embryogenesis is the formation of a plant embryo from a cell other than the product of gametic fusion. The need to recognize the determinants of somatic cell fate has prompted investigations on how endogenous factors of donor tissues can determine the pattern of somatic embryo origin. The undertaking of this study was enabled by the newly developed experimental system of somatic embryogenesis of the tree fern Cyathea delgadii Sternb., in which the embryos are produced in hormone-free medium. The contents of 89 endogenous compounds (such as sugars, auxins, cytokinins, gibberellins, stress-related hormones, phenolic acids, polyamines, and amino acids) and cytomorphological features were compared between two types of explants giving rise to somatic embryos of unicellular or multicellular origin. We found that a large content of maltose, 1-kestose, abscisic acid, biologically active gibberellins, and phenolic acids was characteristic for single-cell somatic embryo formation pattern. In contrast, high levels of starch, callose, kinetin riboside, arginine, and ethylene promoted their multicellular origin. Networks for visualization of the relations between studied compounds were constructed based on the data obtained from analyses of a Pearson correlation coefficient heatmap. Our findings present for the first time detailed features of donor tissue that can play an important role in the somatic-to-embryogenic transition and the somatic embryo origin.

Overexpression of Salicylic Acid Carboxyl Methyltransferase (CsSAMT1) Enhances Tolerance to Huanglongbing Disease in Wanjincheng Orange (Citrus sinensis (L.) Osbeck).

Citrus Huanglongbing (HLB) disease or citrus greening is caused by Candidatus Liberibacter asiaticus (Las) and is the most devastating disease in the global citrus industry. Salicylic acid (SA) plays a central role in regulating plant defenses against pathogenic attack. SA methyltransferase (SAMT) modulates SA homeostasis by converting SA to methyl salicylate (MeSA). Here, we report on the functions of the citrus SAMT (CsSAMT1) gene from HLB-susceptible Wanjincheng orange (Citrus sinensis (L.) Osbeck) in plant defenses against Las infection. The CsSAMT1 cDNA was expressed in yeast. Using in vitro enzyme assays, yeast expressing CsSAMT1 was confirmed to specifically catalyze the formation of MeSA using SA as a substrate. Transgenic Wanjincheng orange plants overexpressing CsSAMT1 had significantly increased levels of SA and MeSA compared to wild-type controls. HLB resistance was evaluated for two years and showed that transgenic plants displayed significantly alleviated symptoms including a lack of chlorosis, low bacterial counts, reduced hyperplasia of the phloem cells, and lower levels of starch and callose compared to wild-type plants. These data confirmed that CsSAMT1 overexpression confers an enhanced tolerance to Las in citrus fruits. RNA-seq analysis revealed that CsSAMT1 overexpression significantly upregulated the citrus defense response by enhancing the transcription of disease resistance genes. This study provides insight for improving host resistance to HLB by manipulation of SA signaling in citrus fruits.

Seed dressing with triflumezopyrim controls brown planthopper populations by inhibiting feeding behavior, fecundity and enhancing rice plant resistance.

Triflumezopyrim (TFM), a novel mesoionic insecticide, has high efficiency at a low dosage, and is mainly used to control hopper species. A previous study demonstrated that seed dressing with TFM effectively controlled rice planthopper populations in mechanically transplanted rice fields; however, mode of action for control was unclear. The study shows that seed dressing with TFM resulted in elevated levels of oxalic acid, flavonoids, phenolic substances, callose and other compounds associated with Nilaparvata lugens resistance in rice plants, and low TFM residue content in rice plant stem and grain. Host choice behavioral experiments showed that N. lugens females prefer feeding on untreated rice plants. Electrical penetration graph (EPG) data showed that seed dressing with TFM at medium and high doses significantly prolonged the non-probing period and inhibited phloem ingestion in N. lugens females. These changes led to a significant decrease in female secretion of honeydew, expression of genes encoding vitellogenin and juvenile hormone acid methyltransferase, body weight and longevity, and significantly influenced several physiological parameters resulting in impaired oocyte growth, fecundity and population. Field survey data showed that seed dressing with TFM was efficacious and relatively durable in protecting rice plants from infestation by planthoppers. This study revealed that seed dressing with TFM enhances rice plant resistance to N. lugens by limiting phloem ingestion and increasing the N. lugens non-probing period; this leads to reduced fecundity of females and lowers N. lugens numbers in the field. © 2021 Society of Chemical Industry.

Spatio-temporal immunolocalization of extensin protein and hemicellulose polysaccharides during olive fruit abscission.

Immunocytochemical and molecular analyses reveal that the disassembly of the cell wall may be mediated by changes in the level and subcellular location of extensin protein and hemicelluloses during olive-fruit abscission. Although cell-wall modification is believed to underlie the changes in organ abscission, information concerning the changes in cell-wall proteins and hemicellulose polysaccharides is still limited. The aim of this work was to analyze the spatio-temporal patterns of the distribution of different extensin proteins and hemicelluloses in the abscission zone (AZ) during natural ripe-fruit abscission in olive (Olea europaea L.). In this study, we employed immunogold labeling in the ripe-fruit AZ during olive AZ cell separation, using an expanded set of monoclonal antibodies that recognize different types of hemicelluloses (LM11, LM15, and LM21), callose (anti-(1,3)-β-D-glucan) and extensin (JIM19) epitopes, and transmission electron microscopy imaging. Our data demonstrate that AZ cell separation was accompanied by a loss of the JIM19 extensin epitopes and a reduction in the detection of the LM15 xyloglucan epitopes in AZ cell walls, whereas AZ cells were found to be enriched with respect to the xylan and callose levels of the cell wall during olive ripe-fruit abscission. By contrast, AZ cell-wall polysaccharide remodeling did not involve mannans. Moreover, in ripe-fruit AZ, quantitative RT-PCR analysis revealed that OeEXT1, OeEXT2, OeXTH9, and OeXTH13 genes were downregulated during abscission, whereas the expression of OeXTH1, OeXTH5, and OeXTH14 genes increased during abscission. Taken together, the results indicate that AZ cell-wall dynamics during olive ripe-fruit abscission involves extensin protein and hemicellulose modifications, as well as related expressed genes. This is the first study available demonstrating temporal degradation of extensin protein and hemicelluloses in the AZ at the subcellular level.

Callose Synthesis Suppresses Cell Death Induced by Low-Calcium Conditions in Leaves.

Despite the importance of preventing calcium (Ca) deficiency disorders in agriculture, knowledge of the molecular mechanisms underlying plant adaptations to low-Ca conditions is limited. In this study, we provide evidence for a crucial involvement of callose synthesis in the survival of Arabidopsis (Arabidopsis thaliana) under low-Ca conditions. A mutant sensitive to low-Ca conditions, low calcium sensitive3 (lcs3), exhibited high levels of cell death in emerging leaves and had defects in its expanding true leaves under low-Ca conditions. Further analyses showed that the causal mutation was located in a putative β-1,3-glucan (callose) synthase gene, GLUCAN SYNTHASE-LIKE10 (GSL10). Yeast complementation assay results showed that GSL10 encodes a functional callose synthase. Ectopic callose significantly accumulated in wild-type plants under low-Ca conditions, but at a low level in lcs3 The low-Ca sensitivity of lcs3 was phenocopied by the application of callose synthase inhibitors in wild-type plants, which resulted in leaf expansion failure, cell death, and reduced ectopic callose levels under low-Ca conditions. Transcriptome analyses showed that the expression of genes related to cell wall and defense responses was altered in both wild-type plants under low-Ca conditions and in lcs3 under normal-Ca conditions, suggesting that GSL10 is required for the alleviation of both cell wall damage and defense responses caused by low Ca levels. These results suggest that callose synthesis is essential for the prevention of cell death under low-Ca conditions and plays a key role in plants' survival strategies under low-Ca conditions.

Cell Wall Modifications in Giant Cells Induced by the Plant Parasitic Nematode Meloidogyne incognita in Wild-Type (Col-0) and the fra2 Arabidopsis thaliana Katanin Mutant.

Meloidogyne incognita is a root knot nematode (RKN) species which is among the most notoriously unmanageable crop pests with a wide host range. It inhabits plants and induces unique feeding site structures within host roots, known as giant cells (GCs). The cell walls of the GCs undergo the process of both thickening and loosening to allow expansion and finally support nutrient uptake by the nematode. In this study, a comparative in situ analysis of cell wall polysaccharides in the GCs of wild-type Col-0 and the microtubule-defective fra2 katanin mutant, both infected with M. incognita has been carried out. The fra2 mutant had an increased infection rate. Moreover, fra2 roots exhibited a differential pectin and hemicellulose distribution when compared to Col-0 probably mirroring the fra2 root developmental defects. Features of fra2 GC walls include the presence of high-esterified pectic homogalacturonan and pectic arabinan, possibly to compensate for the reduced levels of callose, which was omnipresent in GCs of Col-0. Katanin severing of microtubules seems important in plant defense against M. incognita, with the nematode, however, to be nonchalant about this “katanin deficiency” and eventually induce the necessary GC cell wall modifications to establish a feeding site.

Plasma Membrane-Associated Receptor-like Kinases Relocalize to Plasmodesmata in Response to Osmotic Stress.

Plasmodesmata act as key elements in intercellular communication, coordinating processes related to plant growth, development, and responses to environmental stresses. While many of the developmental, biotic, and abiotic signals are primarily perceived at the plasma membrane (PM) by receptor proteins, plasmodesmata also cluster receptor-like activities; whether these two pathways interact is currently unknown. Here, we show that specific PM-located Leu-rich-repeat receptor-like-kinases, Qiān Shŏu kinase (QSK1) and inflorescence meristem kinase2, which under optimal growth conditions are absent from plasmodesmata, rapidly relocate and cluster to the pores in response to osmotic stress. This process is remarkably fast, is not a general feature of PM-associated proteins, and is independent of sterol and sphingolipid membrane composition. Focusing on QSK1, previously reported to be involved in stress responses, we show that relocalization in response to mannitol depends on QSK1 phosphorylation. Loss-of-function mutation in QSK1 results in delayed lateral root (LR) development, and the mutant is affected in the root response to mannitol stress. Callose-mediated plasmodesmata regulation is known to regulate LR development. We found that callose levels are reduced in the qsk1 mutant background with a root phenotype resembling ectopic expression of PdBG1, an enzyme that degrades callose at the pores. Both the LR and callose phenotypes can be complemented by expression of wild-type and phosphomimic QSK1 variants, but not by phosphodead QSK1 mutant, which fails to relocalize at plasmodesmata. Together, the data indicate that reorganization of receptor-like-kinases to plasmodesmata is important for the regulation of callose and LR development as part of the plant response to osmotic stress.

Molecular cloning and functional identification of an apple flagellin receptor MdFLS2 gene

The leucine-rich repeat receptor kinase flagellin-sensing 2 gene (MdFLS2; Gene ID: MDP0000254112) was cloned from Royal Gala apple (Malus×domestica Borkh.). This gene contained a complete open reading frame of 3474 bp that encoded 1158 amino acids. The phylogenetic tree indicated that Prunus persica FLS2 exhibited the highest sequence similarity to MdFLS2. The PlantCare database suggests that the promoter sequence of MdFLS2 contains several typical cis-acting elements, including ethylene-, gibberellin-, salicylic acid-, and drought-responsive elements. Quantitative real-time PCR analysis showed that MdFLS2 was widely expressed in the different tissues of the apple and most highly expressed in the leaves. Furthermore, MdFLS2 was significantly induced by the flagellin elicitor peptide flg22. Treatment of the apple seedling leaves with flg22 resulted in an increase in leaf callose levels with increased treatment duration. An increase in the production of O2– along with the expression of disease-related genes was also observed. An oxidative burst was detected in the treated seedlings, but not in the control seedlings, indicating that flg22 had stimulated the expression of the MdFLS2 gene and its downstream target genes. Furthermore, the ectopic expression of MdFLS2 complemented the function of the Arabidopsis fls2 mutant and conferred enhanced flg22 tolerance to the transgenic Arabidopsis, suggesting that MdFLS2 acts as a positive regulator in the response to pathogens in apple.

Callose-Regulated Symplastic Communication Coordinates Symbiotic Root Nodule Development

The formation of nitrogen-fixing nodules in legumes involves the initiation of synchronized programs in the root epidermis and cortex to allow rhizobial infection and nodule development. In this study, we provide evidence that symplastic communication, regulated by callose turnover at plasmodesmata (PD), is important for coordinating nodule development and infection in Medicago truncatula. Here, we show that rhizobia promote a reduction in callose levels in inner tissues where nodules initiate. This downregulation coincides with the localized expression of M.truncatula β-1,3-glucanase 2 (MtBG2), encoding a novel PD-associated callose-degrading enzyme. Spatiotemporal analyses revealed that MtBG2 expression expands from dividing nodule initials to rhizobia-colonized cortical and epidermal tissues. As shown by the transport of fluorescent molecules invivo, symplastic-connected domains are created in rhizobia-colonized tissues and enhanced in roots constitutively expressing MtBG2. MtBG2-overexpressing roots additionally displayed reduced levels of PD-associated callose. Together, these findings suggest an active role for MtBG2 in callose degradation and in the formation of symplastic domains during sequential nodule developmental stages. Interfering with symplastic connectivity led to drastic nodulation phenotypes. Roots ectopically expressing β-1,3-glucanases (including MtBG2) exhibited increased nodule number, and those expressing MtBG2 RNAi constructs or a hyperactive callose synthase (under symbiotic promoters) showed defective nodulation phenotypes. Obstructing symplastic connectivity appears to block a signaling pathway required for the expression of NODULE INCEPTION (NIN) and its target NUCLEAR FACTOR-YA1 (NF-YA1) in the cortex. We conclude that symplastic intercellular communication is proactively enhanced by rhizobia, and this is necessary for appropriate coordination of bacterial infection and nodule development.

ACR11 modulates levels of reactive oxygen species and salicylic acid-associated defense response in Arabidopsis

The ACT domain (aspartate kinase, chorismate mutase and TyrA), an allosteric effector binding domain, is commonly found in amino acid metabolic enzymes. In addition to ACT domain-containing enzymes, plants have a novel family of ACT domain repeat (ACR) proteins, which do not contain any recognizable catalytic domain. Arabidopsis has 12 ACR proteins, whose functions are largely unknown. To study the functions of Arabidopsis ACR11, we have characterized two independent T-DNA insertion mutants, acr11-2 and acr11-3. RNA gel-blot analysis revealed that the expression of wild-type ACR11 transcripts was not detectable in the acr11 mutants. Interestingly, a lesion-mimic phenotype occurs in some rosette leaves of the acr11 mutants. In addition, high levels of reactive oxygen species (ROS), salicylic acid (SA), and callose accumulate in the mutant leaves when grown under normal conditions. The expression of several SA marker genes and the key SA biosynthetic gene ISOCHORISMATE SYNTHASE1 is up-regulated in the acr11 mutants. Furthermore, the acr11 mutants are more resistant to the infection of bacterial pathogen Pseudomonas syringae pathovar tomato DC3000. These results suggest that ACR11 may be directly or indirectly involved in the regulation of ROS and SA accumulation, which in turn modulates SA-associated defense responses and disease resistance in Arabidopsis.

ANN1 and ANN2 Function in Post-Phloem Sugar Transport in Root Tips to Affect Primary Root Growth.

Annexins are a multigene family of calcium-dependent membrane-binding proteins that play important roles in plant cell signaling. Annexins are multifunctional proteins, and their function in plants is not comprehensively understood. Arabidopsis (Arabidopsis thaliana) annexins ANN1 and ANN2 are 64% identical in their primary structure, and both are highly expressed in seedlings. Here, we showed that ann-mutant seedlings grown in the absence of sugar show decreased primary root growth and altered columella cells in root caps; however, these mutant defects are rescued by Suc, Glc, or Fru. In seedlings grown without sugar, significant up-regulation of photosynthetic gene expression and chlorophyll accumulation was found in ann-mutant cotyledons compared to that in wild type, which indicates potential sugar starvation in the roots of ann-mutant seedlings. Unexpectedly, the overall sugar content of ann-mutant primary roots was significantly higher than that of wild-type roots when grown without sugar. To examine the diffusion of sugar along the entire root to the root tip, we examined the unloading pattern of carboxyfluorescein dye and found that post-phloem sugar transport was impaired in ann-mutant root tips compared to that in wild type. Increased levels of ROS and callose were detected in the root tips of ann-mutant seedlings grown without Suc, the latter of which would restrict plasmodesmal sugar transport to root tips. Our results indicate that ANN1 and ANN2 play an important role in post-phloem sugar transport to the root tip, which in turn indirectly influences photosynthetic rates in cotyledons. This study expands our understanding of the function of annexins in plants.