Effect of gibberellin on crown root development in the mutant of the rice plasmodesmal Germin-like protein OsGER4.

Rice is an essential but highly stress-susceptible crop, whose root system plays an important role in plant development and stress adaptation. The rice root system architecture is controlled by gene regulatory networks involving different phytohormones including auxin, jasmonate, and gibberellin. Gibberellin is generally known as a molecular clock that interacts with different pathways to regulate root meristem development. The exogenous treatment of rice plantlets with Gibberellin reduced the number of crown roots, whilst the exogenous jasmonic acid treatment enhanced them by involving a Germin-like protein OsGER4. Due to those opposite effects, this study aims to investigate the effect of Gibberellin on crown root development in the rice mutant of the plasmodesmal Germin-like protein OsGER4. Under exogenous gibberellin treatment, the number of crown roots significantly increased in osger4 mutant lines and decreased in the OsGER4 overexpressed lines. GUS staining showed that OsGER4 was strongly expressed in rice root systems, particularly crown and lateral roots under GA3 application. Specifically, OsGER4 was strongly expressed from the exodermis, epidermis, sclerenchyma to the endodermis layers of the crown root, along the vascular bundle and throughout LR primordia. The plasmodesmal protein OsGER4 is suggested to be involved in crown root development by maintaining hormone homeostasis, including Gibberillin.

The Germin-like protein OsGER4 is involved in promoting crown root development under exogenous jasmonic acid treatment in rice.

In rice (Oryza sativa L.), crown roots (CRs) have many important roles in processes such as root system expansion, water and mineral uptake, and adaptation to environmental stresses. Phytohormones such as auxin, cytokinin, and ethylene are known to control CR initiation and development in rice. However, the role of jasmonic acid (JA) in CR development remained elusive. Here, we report that JA promotes CR development by regulating OsGER4, a rice Germin-like protein. Root phenotyping analysis revealed that exogenous JA treatment induced an increase in CR number in a concentration-dependent manner. A subsequent genome-wide association study and gene expression analyses pinpointed a strong association between the Germin-like protein OsGER4 and the increase in CR number under exogenous JA treatment. The ProGER4::GUS reporter line showed that OsGER4 is a hormone-responsive gene involved in various stress responses, mainly confined to epidermal and vascular tissues during CR primordia development and to vascular bundles of mature crown and lateral roots. Notable changes in OsGER4 expression patterns caused by the polar auxin transport inhibitor NPA support its connection to auxin signaling. Phenotyping experiments with OsGER4 knockout mutants confirmed that this gene is required for CR development under exogenous JA treatment. Overall, our results provide important insights into JA-mediated regulation of CR development in rice.

Functional divergence of GLP genes between G. barbadense and G. hirsutum in response to Verticillium dahliae infection.

Germin-like proteins (GLPs) play important roles in plant disease resistance but are rarely reported in cotton. We compared the expression of GLPs in Verticillium dahliae inoculate G. hirsutum (susceptible) and G. barbadense (resistant) and enriched 11 differentially expressed GLPs. 2741 GLP proteins identified from 53 species determined that GLP probably originated from algae and could be classified into 7 clades according to phylogenetic analysis, among which Clade I is likely the most ancient. Cotton GLP (two allopolyploids and two diploids) genes within a shared clade were highly conserved. Intriguingly, clade VII genes were mainly located in gene clusters that derived from the expansion of LTR transposons. Clade VII members expressed mainly in root which is the first battle against Verticillium dahlia and could be induced more intensely in G. barbadense than G. hirsutum. The GLP genes are resistant to Verticillium dahliae, which can be further investigated against Verticillium wilt.

ZmGLP1, a Germin-like Protein from Maize, Plays an Important Role in the Regulation of Pathogen Resistance.

A gene encoding a protein similar to germin-like proteins (GLPs) was obtained from maize (Zea mays) and designated as ZmGLP1. Based on the ZmGLP1 conserved domain and phylogenetic status, ZmGLP1 was grouped into GLP subfamily b and has high similarity to OsGLP8-14 from Oryza sativa. ZmGLP1 is expressed in different maize tissues during different growth stages and is mainly expressed in the stems and leaves. The induced expression patterns confirmed that ZmGLP1 is differentially expressed under abiotic and hormone stress; it had an early response to jasmonic acid (JA) and ethephon (ET) but a late response to salicylic acid (SA) and was significantly upregulated under Bipolaris maydis infection. The overexpression of ZmGLP1 in Arabidopsis improved the resistance to biotrophic Pseudomonas syringae pv. tomato DC3000 (PstDC3000) and necrotrophic Sclerotinia sclerotiorum by inducing the expression of JA signaling-related genes. Moreover, the hydrogen peroxide (H2O2) content increased due to the overexpression of ZmGLP1 in Arabidopsis after pathogen infection. Compared to the wild-type control, the H2O2 content of ZmGLP1-overexpressing Arabidopsis infected by PstDC3000 increased significantly but was lower in transgenic plants infected with S. sclerotiorum. Furthermore, high-performance liquid chromatography-tandem mass (HPLC-MS/MS) spectrometry showed that the JA contents of ZmGLP1-overexpressing Arabidopsis markedly increased after pathogen infection. However, the improved resistance of ZmGLP1-overexpressing Arabidopsis pretreated with the JA biosynthetic inhibitor, sodium diethyldithiocarbamate trihydrate (DIECA), was suppressed. Based on these findings, we speculate that ZmGLP1 plays an important role in the regulation of Arabidopsis resistance to biotrophic PstDC3000 and necrotrophic S. sclerotiorum; the regulatory effects are achieved by inducing plant oxidative burst activity and activation of the JA signaling pathway.

Overexpression of germin-like protein GmGLP10 enhances resistance to Sclerotinia sclerotiorum in transgenic tobacco.

Germin-like proteins (GLPs) are ubiquitous water-soluble glycoproteins that are located in the extracellular matrix. These proteins have been reported to play vital roles in diverse biological processes. In the present study, a GLP in soybean (Glycine max L. Merr.), GmGLP10, was characterized. Sequence analysis revealed that the GmGLP10 gene (GenBank Accession Number EU916258) encodes a 213-amino acid (aa) protein, which contains a N-terminal signal peptide at 1-22 aa and is highly homologous to the members of the GER2 subfamily. GmGLP10 was highly expressed in the leaves, but very faint in the roots. The expression of GmGLP10 was induced by methyl jasmonate (MeJA), ethylene (ET), salicylic acid (SA), oxalate acid (OA), and the infection of Sclerotinia sclerotiorum. Overexpression of GmGLP10 in transgenic tobacco significantly enhanced tolerance to OA and S. sclerotiorum infection. Moreover, higher levels of H2O2 and the upregulated expression of a set of plant defense-related genes and HR (hypersensitive response)-associated genes were detected in the transgenic plants. These results suggest that GmGLP10 functions as a positive regulator of resistance to S. sclerotiorum.

Proteomic discovery of H2O2 response in roots and functional characterization of PutGLP gene from alkaligrass.

MAIN CONCLUSION: Hydrogen peroxide-responsive pathways in roots of alkaligrass analyzed by proteomic studies and PutGLP enhance the plant tolerance to saline-, alkali- and cadmium-induced oxidative stresses. Oxidative stress adaptation is critical for plants in response to various stress environments. The halophyte alkaligrass (Puccinellia tenuiflora) is an outstanding pasture with strong tolerance to salt and alkali stresses. In this study, iTRAQ- and 2DE-based proteomics approaches, as well as qRT-PCR and molecular genetics, were employed to investigate H2O2-responsive mechanisms in alkaligrass roots. The evaluation of membrane integrity and reactive oxygen species (ROS)-scavenging systems, as well as abundance patterns of H2O2-responsive proteins/genes indicated that Ca2+-mediated kinase signaling pathways, ROS homeostasis, osmotic modulation, and transcriptional regulation were pivotal for oxidative adaptation in alkaligrass roots. Overexpressing a P. tenuiflora germin-like protein (PutGLP) gene in Arabidopsis seedlings revealed that the apoplastic PutGLP with activities of oxalate oxidase and superoxide dismutase was predominantly expressed in roots and played important roles in ROS scavenging in response to salinity-, alkali-, and CdCl2-induced oxidative stresses. The results provide insights into the fine-tuned redox-responsive networks in halophyte roots.

Identification of manganese efficiency candidate genes in winter barley (Hordeum vulgare) using genome wide association mapping.

BACKGROUND: Manganese (Mn) has several essential functions in plants, including a role as cofactor in the oxygen evolving complex (OEC) of photosystem II (PSII). Manganese deficiency is a major plant nutritional disorder in winter cereals resulting in significant yield reductions and winter kill in more severe cases. Among the winter cereals, genotypes of winter barley are known to differ considerably in tolerance to Mn deficiency, but the genes controlling the Mn deficiency trait remains elusive. RESULTS: Experiments were conducted using 248 barley varieties, cultivated in six distinct environments prone to induce Mn deficiency. High-throughput phenotyping for Mn deficiency was performed by chlorophyll a (Chl a) fluorescence analysis to quantify the quantum yield efficiency of PSII. High-throughput phenotyping in combination with ICP-OES based multi-element analyses allowed detection of marker-trait associations by genome wide association (GWA) mapping. Several key candidate genes were identified, including PSII subunit proteins, germin like proteins and Mn superoxide dismutase. The putative roles of the encoded proteins in Mn dependent metabolic processes are discussed. CONCLUSIONS: Fifty-four candidate genes were identified by Chl a fluorescence phenotyping and association genetics. Tolerance of plants to Mn deficiency, which is referred to as Mn efficiency, appeared to be a complex trait involving many genes. Moreover, the trait appeared to be highly dependent on the environmental conditions in field. This study provides the basis for an improved understanding of the parameters influencing Mn efficiency and is valuable in future plant breeding aiming at producing new varieties with improved tolerance to cultivation in soil prone to induce Mn deficiency.

Functional characterization of germin and germin-like protein genes in various plant species using transgenic approaches.

The important role of germins (GER) and genes coding for germin-like proteins (GLP) in responses against various stresses in both homologous and heterologous systems is well validated. This review summarizes the work on their functional validation using various biotechnological approaches. The genes are widely expressed during a specific period of plant growth and development, and exhibit a pattern of evolutionary subfunctionalization at both the intracellular and whole plant level. Their applications against various biotic and abiotic stresses, especially against fungal pathogens, are enormous. Although the validation of these proteins against various stresses has led to the development of commercially and agronomically important transgenic plants, much work is still needed to exploit this ever-expanding repertoire of genes and deploy them for commercial use. Historical progress of genetic engineering in GERs and GLPs is reviewed, and future prospects for their potential role in crop improvement are highlighted.

In vitro and in vivo evidence for oxalate oxidase activity of a germin-like protein from azalea.

Germins and germin-like proteins (GLPs) comprise large families of extracellular plant glycoproteins that are structurally similar, yet they have been reported to have distinct biochemical activities: oxalate oxidase and superoxide dismutase activities, respectively. We expressed an azalea GLP (RmGLP2) in cultured cells of tobacco, and determined that the extracellular protein fraction and the recombinant RmGLP2 protein purified from these cells catalyzed the oxidation of oxalate. Notably, this activity is purportedly restricted to germin and has not been demonstrated for a GLP. Although the specific activity of the purified RmGLP2 protein was low compared with that of a previously characterized barley germin/oxalate oxidase, tobacco cells expressing RmGLP2 exhibited significantly reduced oxalate levels. Thus, RmGLP2 represents the first reported GLP with oxalate oxidase activity.

Sunflower germin-like protein HaGLP1 promotes ROS accumulation and enhances protection against fungal pathogens in transgenic Arabidopsis thaliana.

KEY MESSAGE: The novel sunflower gene HaGLP1 is the first germin-like protein characterized from the family Asteraceae. It alters the host redox status and confers protection against Sclerotinia sclerotiorum and Rhizoctonia solani. Germin-like proteins (GLPs) are a large, diverse and ubiquitous family of plant glycoproteins belonging to the Cupin super family. These proteins have been widely studied because of their diverse roles in important plant processes, including defence. The novel sunflower gene HaGLP1 encodes the first germin-like protein characterized from the family Asteraceae. To analyse whether constitutive in vivo expression of the HaGLP1 gene may lead to disease tolerance, we developed transgenic Arabidopsis plants that were molecularly characterized and biologically assessed after inoculation with Sclerotinia sclerotiorum or Rhizoctonia solani. HaGLP1 expression in Arabidopsis plants conferred tolerance to S. sclerotiorum at the first stages of disease and interfered with R. solani infection, thus giving rise to significant protection against the latter. Furthermore, HaGLP1 expression in Arabidopsis plants elevated endogenous ROS levels. HaGLP1-induced tolerance does not appear to be related to a constitutive induction of the plant defence or the ROS-related genes examined here. In conclusion, our data suggest that HaGLP1 is an interesting candidate for the engineering of plants with increased fungal tolerance and that this gene could also be useful for the selection of naturally overexpressing sunflower genotypes for conventional breeding purposes.

Characterization of germin-like protein with polyphenol oxidase activity from Satsuma mandarine.

Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris-HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65°C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity.

Characteristics of Recombinant Chlamydomonas reinhardtii Expressing Putative Germin-Like Protein from Neopyropia yezoensis.

Since microalgae face various environmental stresses for the high production of biofuels, multiple studies have been performed to determine if microalgae are resistant to these various stresses. In this study, the viability of cells under various abiotic stresses was investigated by introducing a putative germin-like protein (GLP) from Neopyropia yezoensis, which was known to be related in the resistance to abiotic stresses. The expression of GLP in Chlamydomonas reinhardtii allowed cells to grow better in various abiotic stress environments. In nitrogen starvation conditions, recombinant cells accumulated the lipid droplet 1.46-fold more than wild-type cells and responded more rapidly to form palmelloid forms. Under high-temperature, hydrogen peroxide conditions and saline stress, the survival rate was increased 3.5 times, 2.19 times, and 3.19 times in recombinant C. reinhardtii with GLP, respectively. The expression level of genes related to pathways in response to various stresses increased 2-fold more under those conditions. This result will be useful for the development of microalgae that can grow better and produce more biofuels under different stress conditions.

OsGLP8-7 interacts with OsPRX111 to detoxify excess copper in rice.

Lignin is a phenolic biopolymer generated from phenylpropanoid pathway in the secondary cell wall and is required for defense of plants against various stress. Although the fact of stress-induced lignin deposition has been clearly demonstrated, it remains largely elusive how the formation of lignin is promoted under Cu stress. The present study showed that OsGLP8-7, an extracellular glycoprotein of rice (Oryza sativa L.), plays an important function against Cu stress. The loss function of OsGLP8-7 results in Cu sensitivity whereas overexpression of OsGLP8-7 scavenges Cu-induced superoxide anion (O2•-). OsGLP8-7 interacts with apoplastic peroxidase111 (OsPRX111) and elevates OsPRX111 stability when exposed to excess Cu. In OsGLP8-7 overexpressing (OE) lines, the retention of Cu within cell wall limiting Cu uptake into cytoplasm is attributed to the enhanced lignification required for Cu tolerance. Exogenous application of a lignin inhibitor can impair the Cu tolerance of transgenic Arabidopsis lines overexpressing OsGLP8-7. In addition, co-expression of OsGLP8-7 and OsPRX111 genes in tobacco leaves leads to an improved lignin deposition compared to leaves expressing each gene individually or the empty vector. Taken together, our findings provided the convincing evidences that the interaction between OsGLP8-7 and OsPRX111 facilitates effectively lignin polymerization, thereby contributing to Cu tolerance in rice.

A Germin-Like Protein GLP1 of Legumes Mediates Symbiotic Nodulation by Interacting with an Outer Membrane Protein of Rhizobia.

Rhizobia can infect legumes and induce the coordinated expression of symbiosis and defense genes for the establishment of mutualistic symbiosis. Numerous studies have elucidated the molecular interactions between rhizobia and host plants, which are associated with Nod factor, exopolysaccharide, and T3SS effector proteins. However, there have been relatively few reports about how the host plant recognizes the outer membrane proteins (OMPs) of rhizobia to mediate symbiotic nodulation. In our previous work, a gene (Mhopa22) encoding an OMP was identified in Mesorhizobium huakuii 7653R, whose homologous genes are widely distributed in Rhizobiales. In this study, a germin-like protein GLP1 interacting with Mhopa22 was identified in Astragalus sinicus. RNA interference of AsGLP1 resulted in a decrease in nodule number, whereas overexpression of AsGLP1 increased the number of nodules in the hairy roots of A. sinicus. Consistent symbiotic phenotypes were identified in Medicago truncatula with MtGLPx (refer to medtr7g111240.1, the isogeny of AsGLP1) overexpression or Tnt1 mutant (glpx-1) in symbiosis with Sinorhizobium meliloti 1021. The glpx-1 mutant displayed hyperinfection and the formation of more infection threads but a decrease in root nodules. RNA sequencing analysis showed that many differentially expressed genes were involved in hormone signaling and symbiosis. Taken together, AsGLP1 and its homology play an essential role in mediating the early symbiotic process through interacting with the OMPs of rhizobia. IMPORTANCE This study is the first report to characterize a legume host plant protein to sense and interact with an outer membrane protein (OMP) of rhizobia. It can be speculated that GLP1 plays an essential role to mediate early symbiotic process through interacting with OMPs of rhizobia. The results provide deeper understanding and novel insights into the molecular interactive mechanism of a legume symbiosis signaling pathway in recognition with rhizobial OMPs. Our findings may also provide a new perspective to improve the symbiotic compatibility and nodulation of legume.

OsGLP3-7 positively regulates rice immune response by activating hydrogen peroxide, jasmonic acid, and phytoalexin metabolic pathways.

Although germin-like proteins (GLPs) have been demonstrated to participate in plant biotic stress responses, their specific functions in rice disease resistance are still largely unknown. Here, we report the identification and characterization of OsGLP3-7, a member of the GLP family in rice. Expression of OsGLP3-7 was significantly induced by pathogen infection, jasmonic acid (JA) treatment, and hydrogen peroxide (H2 O2 ) treatment. OsGLP3-7 was highly expressed in leaves and sublocalized in the cytoplasm. Overexpression of OsGLP3-7 increased plant resistance to leaf blast, panicle blast, and bacterial blight, whereas disease resistance in OsGLP3-7 RNAi silenced plants was remarkably compromised, suggesting this gene is a positive regulator of disease resistance in rice. Further analysis showed that OsGLP3-7 has superoxide dismutase (SOD) activity and can influence the accumulation of H2 O2 in transgenic plants. Many genes involved in JA and phytoalexin biosynthesis were strongly induced, accompanied with elevated levels of JA and phytoalexins in OsGLP3-7-overexpressing plants, while expression of these genes was significantly suppressed and the levels of JA and phytoalexins were reduced in OsGLP3-7 RNAi plants compared with control plants, both before and after pathogen inoculation. Moreover, we showed that OsGLP3-7-dependent phytoalexin accumulation may, at least partially, be attributed to the elevated JA levels observed after pathogen infection. Taken together, our results indicate that OsGLP3-7 positively regulates rice disease resistance by activating JA and phytoalexin metabolic pathways, thus providing novel insights into the disease resistance mechanisms conferred by GLPs in rice.

Genome-wide identification and functional analysis of cupin_1 domain-containing members involved in the responses to Sclerotinia sclerotiorum and abiotic stress in Brassica napus.

Cupin_1 domain-containing proteins (CDPs) are ubiquitously present in higher plants, which are known to play essential roles in various biological processes. In this study, we carried out genome-wide characterization and systematic investigation of the CDP genes in Brassica napus. A total of 96 BnCDPs, including 71 germin-like proteins (GLPs; proteins with a single cupin_1 domain) and 25 CDP bicupins (proteins with two cupin_1 domains), were identified and clustered into six distinct subfamilies (I-VI) based on the phylogenic analysis, gene structure and motif distribution. Further analysis indicated that whole-genome duplication (WGD) and segmental duplication are main contributors to the species-specific expansion of the BnCDP gene family, and all the duplicated genes subsequently underwent strong purification selection. The promoter region of BnCDPs showed enrichment of cis-regulatory elements associated with development, hormone and stress, as well as transcription factor binding sites, which validates the prediction that BnCDPs are widely involved in plant growth and biotic and abiotic stress responses. The BnCDPs in different subfamilies exhibited obvious differences in expression among 30 developmental tissues/stages of B. napus, implying that BnCDPs may be involved in tissue- and stage-specific developmental processes. Similar trends in expression of most BnCDPs were observed under Sclerotinia sclerotiorum inoculation and four abiotic stresses (dehydration, cold, ABA and salinity), particularly the BnGLPs in subfamily I and III with single cupin_1 domain, revealing that BnCDPs are of great importance in the environmental adaption of B. napus. We then performed a genome-wide association study (GWAS) of 274 B. napus core germplasms on S. sclerotiorum resistance and identified four significantly associated loci harboring five BnGLPs. The expression levels of two candidate genes, BnGLP1.A08 and BnGLP1.C08, were significantly correlated with S. sclerotiorum resistance. Their functional responses to multiple stages of S. sclerotiorum inoculation and four abiotic stresses were further examined through qPCR. Overall, this study provides rich resources for research on the function and evolutionary playground of CDP genes.

Wheat germin-like protein: Studies on chitin/chitosan matrix for tissue engineering applications.

Advances in tissue engineering require the development of new biomaterials with adequate properties of cell attachment and growth. The properties of biomaterials can be improved by incorporation of bioactive molecules to enhance in vitro and/or in vivo functions. In this work, we study the role of a wheat germin-like protease inhibitor (GLPI), free or immobilized in biocompatible matrices to improve cell-attachment ability on different mammalian cell lines. The phylogenetic relationships and functional diversity of the GLPI were analyzed among diverse genera to get insights into sequence motif conservations. The cytocompatibility effect of free GLPI on C2C12 premyoblastic cells and B16 cells as tumoral model has been tested. GLPI promoted proliferation and metabolic activity of both cell types on in vitro models, not showing cytotoxic effects. Furthermore, GLPI was immobilized in chitin microparticles and in chitosan films; we demonstrated an accelerated cell adhesion process in both biomaterials.

GhABP19, a Novel Germin-Like Protein From Gossypium hirsutum, Plays an Important Role in the Regulation of Resistance to Verticillium and Fusarium Wilt Pathogens.

Germin-like proteins (GLPs) are water-soluble plant glycoproteins belonging to the cupin superfamily. The important role of GLPs in plant responses against various abiotic and biotic stresses, especially pathogens, is well validated. However, little is known about cotton GLPs in relation to fungal pathogens. Here, a novel GLP gene was isolated from Gossypium hirsutum and designated as GhABP19. The expression of GhABP19 was upregulated in cotton plants inoculated with Verticillium dahliae and Fusarium oxysporum and in response to treatment with jasmonic acid (JA) but was suppressed in response to salicylic acid treatment. A relatively small transient increase in GhABP19 was seen in H2O2 treated samples. The three-dimensional structure prediction of the GhABP19 protein indicated that the protein has three histidine and one glutamate residues responsible for metal ion binding and superoxide dismutase (SOD) activity. Purified recombinant GhABP19 exhibits SOD activity and could inhibit growth of V. dahliae, F. oxysporum, Rhizoctonia solani, Botrytis cinerea, and Valsa mali in vitro. To further verify the role of GhABP19 in fungal resistance, GhABP19-overexpressing Arabidopsis plants and GhABP19-silenced cotton plants were developed. GhABP19-transgenic Arabidopsis lines showed much stronger resistance to V. dahliae and F. oxysporum infection than control (empty vector) plants did. On the contrary, silencing of GhABP19 in cotton conferred enhanced susceptibility to fungal pathogens, which resulted in necrosis and wilt on leaves and vascular discoloration in GhABP19-silenced cotton plants. The H2O2 content and endogenous SOD activity were affected by GhABP19 expression levels in Arabidopsis and cotton plants after inoculation with V. dahliae and F. oxysporum, respectively. Furthermore, GhABP19 overexpression or silencing resulted in activation or suppression of JA-mediated signaling, respectively. Thus, GhABP19 plays important roles in the regulation of resistance to verticillium and fusarium wilt in plants. These modulatory roles were exerted by its SOD activity and ability to activate the JA pathway. All results suggest that GhABP19 was involved in plant disease resistance.

Functional characterization of the rice root Germin-like protein gene-1 (OsRGLP1) promoter in Nicotiana tabacum.

Germin (GER) and germin-like protein (GLP) genes play a very important role against various stresses. Promoter analysis provide significant insight into gene's function and regulation. Presently, upstream region (1228 bp) of the OsRGLP1 gene was functionally characterized via heterologous expression. It was fused with the Glucuronidase (GUS) reporter gene and the expression cassette was used to transform Nicotiana tabacum using Agrobacterium-mediated transformation. Transgenic plants were examined via quantitative real-time PCR (qPCR) to analyze its role in wounding, salinity, drought, abscisic acid (ABA) and circadian rhythm. OsRGLP1 was highly induced by ABA and drought by showing 28- and 25-fold changes in GUS mRNA level respectively as compared to wounding (fourfold change) and salinity (threefold change). However, no activity was observed in circadian rhythm. Histochemically, strong GUS activity was observed in leaf veins, midrib, epidermal hair, stomata guard cells, stem cortex, root hairs, xylem and phloem and at cellular level in cell wall, cytoplasm and its periphery. OsRGLP1 promoter can be used to develop agronomically important transgenic plants in future food program.

Inactivation, aggregation, secondary and tertiary structural changes of germin-like protein in Satsuma mandarine with high polyphenol oxidase activity induced by ultrasonic processing.

The inhibition of Polyphenol oxidase (PPO) in plants has been widely researched for their important roles in browning reaction. A newly found germin-like protein (GLP) with high PPO activity in Satsuma mandarine was inactivated by low-frequency high-intensity ultrasonic (20 kHz) processing. The effects of ultrasound on PPO activity and structure of GLP were investigated using dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM), circular dichroism (CD) spectral measurement and fluorescence spectral measurement. The lowest PPO activity achieved was 27.4% following ultrasonication for 30 min at 400 W. DLS analysis showed ultrasound caused both aggregation and dissociation of GLP particles. TEM images also demonstrated protein aggregation phenomena. CD spectra exhibited a certain number of loss in α-helix structure content. Fluorescence spectra showed remarkable increase in fluorescence intensity with tiny blue-shift following ultrasonication. In conclusion, ultrasound applied in this study induced structural changes of GLP and eventually inactivated PPO activity.