Identification and Expression Analysis of the PIN and AUX/LAX Gene Families in Ramie (Boehmeria nivea L. Gaud)

Auxin regulates diverse aspects of growth and development. Furthermore, polar auxin transport, which is mediated by the PIN-FORMED (PIN) and AUXIN1/LIKE-AUX (AUX/LAX) proteins, plays a crucial role in auxin distribution. In this study, six PIN and four AUX/LAX genes were identified in ramie (Boehmeria nivea L.). We used qRT-PCR to characterize and analyze the two gene families, including phylogenetic relationships, intron/exon structures, cis-elements, subcellular localization, and the expression patterns in different tissues. The expression of these genes in response to indole-3-acetic acid (IAA) treatment and drought stress was also assessed; the results indicate that most of the BnAUX/LAX and BnPIN genes were regulated as a result of IAA treatment and drought stress. Our study provides insights into ramie auxin transporters and lays the foundation for further analysis of their biological functions in ramie fiber development and adaptation to environmental stresses

Identification and Expression of SAUR Genes in the CAM Plant Agave

Agave species are important crassulacean acid metabolism (CAM) plants and widely cultivated in tropical areas for producing tequila spirit and fiber. The hybrid H11648 of Agave ((A. amaniensis × A. angustifolia) × A. amaniensis) is the main cultivar for fiber production in Brazil, China, and African countries. Small Auxin Up-regulated RNA (SAUR) genes have broad effect on auxin signaling-regulated plant growth and development, while only few SAUR genes have been reported in Agave species. In this study, we identified 43, 60, 24, and 21 SAUR genes with full-length coding regions in A. deserti, A. tequilana, A. H11648, and A. americana, respectively. Although phylogenetic analysis revealed that rice contained a species-specific expansion pattern of SAUR gene, no similar phenomena were observed in Agave species. The in silico expression indicated that SAUR genes had a distinct expression pattern in A. H11648 compared with other Agave species; and four SAUR genes were differentially expressed during CAM diel cycle in A. americana. Additionally, an expression analysis was conducted to estimate SAUR gene expression during different leaf developmental stages, abiotic and biotic stresses in A. H11648. Together, we first characterized the SAUR genes of Agave based on previously published transcriptome datasets and emphasized the potential functions of SAUR genes in Agave’s leaf development and stress responses. The identification of which further expands our understanding on auxin signaling-regulated plant growth and development in Agave species

Transcript profiling reveals auxin and cytokinin signaling pathways and transcription regulation during in vitro organogenesis of Ramie (Boehmeria nivea L. Gaud).

In vitro organogenesis, one of the most common pathways leading to in vitro plant regeneration, is widely used in biotechnology and the fundamental study of plant biology. Although previous studies have constructed a complex regulatory network model for Arabidopsis in vitro organogenesis, no related study has been reported in ramie. To generate more complete observations of transcriptome content and dynamics during ramie in vitro organogenesis, we constructed a reference transcriptome library and ten digital gene expression (DGE) libraries for illumina sequencing. Approximately 111.34 million clean reads were obtained for transcriptome and the DGE libraries generated between 13.5 and 18.8 million clean reads. De novo assembly produced 43,222 unigenes and a total of 5,760 differentially expressed genes (DEGs) were filtered. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database, 26 auxin related and 11 cytokinin related DEGs were selected for qRT-PCR validation of two ramie cultivars, which had high (Huazhu No. 5) or extremely low (Dazhuhuangbaima) shoot regeneration abilities. The results revealed differing regulation patterns of auxin and cytokinin in different genotypes. Here we report the first genome-wide gene expression profiling of in vitro organogenesis in ramie and provide an overview of transcription and phytohormone regulation during the process. Furthermore, the auxin and cytokinin related genes have distinct expression patterns in two ramie cultivars with high or extremely low shoot regeneration ability, which has given us a better understanding of the in vitro organogenesis mechanism. This result will provide a foundation for future phytohormone research and lead to improvements of the ramie regeneration system

Structural Characterization of a Neutralizing Nanobody With Broad Activity Against SARS-CoV-2 Variants

International audienceSARS-CoV-2 and its variants, such as the Omicron continue to threaten public health. The virus recognizes the host cell by attaching its Spike (S) receptor-binding domain (RBD) to the host receptor, ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here, we report the isolation and biological and structural characterization of a single-chain antibody (nanobody) from RBD-immunized alpaca. The nanobody, named DL28, binds to RBD tightly with a K-D of 1.56 nM and neutralizes the original SARS-CoV-2 strain with an IC50 of 0.41 mu g mL(-1). Neutralization assays with a panel of variants of concern (VOCs) reveal its wide-spectrum activity with IC50 values ranging from 0.35 to 1.66 mu g mL(-1) for the Alpha/Beta/Gamma/Delta and an IC50 of 0.66 mu g mL(-1) for the currently prevalent Omicron. Competition binding assays show that DL28 blocks ACE2-binding. However, structural characterizations and mutagenesis suggest that unlike most antibodies, the blockage by DL28 does not involve direct competition or steric hindrance. Rather, DL28 may use a "conformation competition" mechanism where it excludes ACE2 by keeping an RBD loop in a conformation incompatible with ACE2-binding

Isolation, characterization, and structure-based engineering of a neutralizing nanobody against SARS-CoV-2

International audienceSARS-CoV-2 engages with human cells through the binding of its Spike receptor-binding domain (S-RBD) to the receptor ACE2. Molecular blocking of this engagement represents a proven strategy to treat COVID-19. Here, we report a single-chain antibody (nanobody, DL4) isolated from immunized alpaca with picomolar affinity to RBD. DL4 neutralizes SARS-CoV-2 pseudoviruses with an IC50 of 0.101 mu g mL-1 (6.2 nM). A crystal structure of the DL4-RBD complex at 1.75-angstrom resolution unveils the interaction detail and reveals a direct competition mechanism for DL4's ACE2-blocking and hence neutralizing activity. The structural information allows us to rationally design a mutant with higher potency. Our work adds diversity of neutralizing nanobodies against SARS-CoV-2 and should encourage protein engineering to improve antibody affinities in general

Distribution of the assembled unigenes across the KOG categories.

<p>Distribution of the assembled unigenes across the KOG categories.</p

Regulated transcription factors during in vitro organogenesis.

<p>*Subgroup of zinc finger family.</p><p>Regulated transcription factors during in vitro organogenesis.</p

Expression pattern and correlations for auxin and cytokinin related genes in two ramie cultivars, H5 and DZ.

<p>The relative expression level was obtained by qRT-PCR after logarithmic transformation of the data. The correlation coefficient (R) was calculated by SPSS.</p

Differentially expressed genes during in vitro organogenesis were clustered by the k-Means method, which is based on their expression modulation.

<p>The relative expression level was obtained after taking equation and logarithmic transformations of RPKM.</p