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.

The germin-like protein OsGLP8-7 is involved in lignin synthesis for acclimation to copper toxicity in rice.

Although copper (Cu) is an essential microelement for plant growth and development, excess Cu results in a dramatic reduction in crop yield and quality. In the present study, we report that rice germin-like protein 8-7 (OsGLP8-7) plays a crucial role against Cu toxicity. The results showed that the transcriptional expression of the OsGLP8-7 gene was remarkably upregulated in the root and leaf by Cu treatment. The depletion of OsGLP8-7 significantly decreased the elongation of the primary root and plant height of rice under excess Cu. This hypersensitivity of osglp8-7 mutants towards excess Cu may be attributed to the weaker Cu retention in the cell wall compared with wild-type rice (Dongjin, DJ). Consistently, Cu-induced phenylpropanoid biosynthesis was compromised in osglp8-7 mutants based on RNA-Seq and qRT-PCR analysis. Furthermore, osglp8-7 mutants displayed a reduction of lignin deposition in the cell wall, and subsequently altered cell morphology. Osglp8-7 mutant lines also had higher Cu-induced O2•- and H2O2 levels than those of DJ under Cu stress. The results suggest that OsGLP8-7 participates in lignin synthesis for the acclimation to excess Cu. These findings provide a better understanding of a novel mechanism of germin-like proteins in the alleviation of heavy metal toxicity in rice.

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.

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.

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.

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.

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.

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.