Ectopic expression of Atleafy in Brassica juncea cv. Geeta for early flowering

High temperature stress during pod filling severely affects the yield of Brassica juncea. Early flowering can evade the terminal heat stress and result in early maturity of the crop. In this study, a regeneration and transformation protocol has been standardized for B. juncea cv. Geeta. Hypocotyl from 5-day-old seedlings were used as explants. Of the various combinations of auxins and cytokinins tried along with Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) medium, MS + IAA (0.2 mg/l) + BA (3 mg/l) proved best for shoot regeneration with 89.9 % regeneration efficiency. To induce early flowering Leafy gene from Arabidopsis thaliana was transformed using Agrobacterium mediated transformation method. After 12 weeks transgenic plants showed flowering in vitro whereas their untransformed counterpart did not flower even after 16 weeks. The maximum transformation frequency was 4 %.     

Recovery of dicer-like 1-late flowering phenotype by miR172 expressed by the noncanonical DCL4-dependent biogenesis pathway

MicroRNAs (miRNAs) act as down-regulators of gene expression, and play a dominant role in eukaryote development. In Arabidopsis thaliana, DICER-LIKE 1 (DCL1) is the main processor in miRNA biogenesis, and dcl1 mutants show various developmental defects at the early stage of embryogenesis or at gamete formation. However, miRNAs responsible for the respective developmental stages of the dcl1 defects have not been identified. Here, we developed a DCL1-independent miRNA expression system using the unique DCL4-dependent miRNA, miR839. By replacing the mature sequence in the miR839 precursor sequence with that of miR172, one of the most widely conserved miRNAs in angiosperms, we succeeded in expressing miR172 from a chimeric miR839 precursor in dcl1-7 plants and observed the repression of miR172 target gene expression. In parallel, the DCL4-dependent miR172 expression rescued the late flowering phenotype of dcl1-7 by acceleration of flowering. We established the DCL1-independent miRNA expression system, and revealed that the reduction of miR172 expression is responsible for the dcl1-7 late flowering phenotype.     

Analysis of biochemical variations and microRNA expression in wild (Ipomoea campanulata) and cultivated (Jacquemontia pentantha) species exposed to in vivo water stress

The current study analyses few important biochemical parameters and microRNA expression in two closely related species (wild but tolerant Ipomoea campanulata L. and cultivated but sensitive Jacquemontia pentantha Jacq.G.Don) exposed to water deficit conditions naturally occurring in the field. Under soil water deficit, both the species showed reduction in their leaf area and SLA as compared to well-watered condition. A greater decrease in chlorophyll was noticed in J. pentantha (~50 %) as compared to I. campanulata (20 %) under stress. By contrast, anthocyanin and MDA accumulation was greater in J. pentantha as compared to I. campanulata. Multiple isoforms of superoxide dismutases (SODs) with differing activities were observed under stress in these two plant species. CuZnSOD isoforms showed comparatively higher induction (~10–40 %) in I. campanulata than J. pentantha. MicroRNAs, miR398, miR319, miR395 miR172, and miR408 showed opposing expression under water deficit in these two plant species. Expression of miR156, miR168, miR171, miR172, miR393, miR319, miR396, miR397 and miR408 from either I. campanulata or J. pentantha or both demonstrated opposite pattern of expression to that of drought stressed Arabidopsis. The better tolerance of the wild species (I. campanulata) to water deficit could be attributed to lesser variations in chlorophyll and anthocyanin levels; and relatively higher levels of SODs than J. pentantha. miRNA expression was different in I. campanulata than J. pentantha.     

The identification of microRNAs in the whitespotted bamboo shark (Chiloscyllium plagiosum) liver by Illumina sequencing

MicroRNAs are indispensable players in the regulation of a broad range of biological processes. Here, we report the first deep sequencing of the whitespotted bamboo shark (Chiloscyllium plagiosum) liver. We mapped 91 miRNAs in the Callorhinchus milii genome that have previously been described in the Danio rerio, Fugu rubripes, Oryzias latipes, Xenopus laevis, Xenopus tropicalis, Homo sapiens, and Mus musculus. In addition, 156 new putative candidate (PC) C. plagiosum miRNAs were identified. From these 247 miRNAs, 39 miRNA clusters were identified, and the expression of these clustered miRNAs was observed to vary significantly. A total of 7 candidate miRNAs were selected for expression confirmation by stem-loop RT-PCR. This study resulted in the addition of a significant number of novel miRNA sequences to GenBank and laid the foundation for further understanding of the function of miRNAs in the regulation of C. plagiosum liver development.     

Profiling of MicroRNAs under Wound Treatment in Aquilaria sinensis to Identify Possible MicroRNAs Involved in Agarwood Formation

Agarwood, a kind of highly valued non-timber product across Asia, is formed only when its resource trees -- the endangered genus Aquilaria are wounded or infected by some microbes. To promote the efficiency of agarwood production and protect the wild resource of Aquilaria species, we urgently need to reveal the regulation mechanism of agarwood formation. MicroRNAs (miRNAs) are a group of gene expression regulators with overwhelming effects on a large spectrum of biological processes. However, their roles in agarwood formation remain unknown. This work aimed at identifying possible miRNAs involved in the wound induced agarwood formation. In this study, the high-throughput sequencing was adopted to identify miRNAs and monitor their expression under wound treatment in the stems of A. sinensis. The miR171, miR390, miR394, miR2111, and miR3954 families remained at the reduced level two days after the treatment. 131 homologous miRNAs in the 0.5 h library showed over three-fold variation of read number compared with the control library, of which 12 exhibiting strong expression alterations were further confirmed by real-time quantitative PCR. Target prediction and annotation of the miRNAs demonstrated that the binding, metabolic process, catalytic activity, and cellular process are the most common functions of the predicted targets of these newly identified miRNAs in A.sinensis. The cleaveage sites of three newly predicted targets were verified by 5''RACE.     

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.     

A subset of <Emphasis Type="Italic">OsSERK</Emphasis> genes,including <Emphasis Type="Italic">OsBAK1</Emphasis>, affects normal growth and leaf development of rice

Since the identification of BRI1-Associated receptor Kinase 1 (BAK1), a member of the Somatic Embryogenesis Receptor Kinase (SERK) family, the dual functions of BAK1 in BR signaling and innate immunity in Arabidopsis have attracted considerable attention as clues for understanding developmental processes that must be balanced between growth and defense over the life of plants. Here, we extended our research to study cellular functions of OsSERKs in rice. As it was difficult to identify an authentic ortholog of AtBAK1 in rice, we generated transgenic rice in which the expression of multiple OsSERK genes, including OsBAK1, was reduced by OsBAK1 RNA interference. Resulting transgenic rice showed reduced levels of Os-BAK1 and decreased sensitivity to BL, leading to semidwarfism in overall growth. Moreover, they resulted in abnormal growth patterns, especially in leaf development. Most of the OsBAK1RNAi transgenic rice plants were defective in the development of bulliform cells in the leaf epidermal layer. They also showed increased expression level of pathogenesis-related gene and enhanced susceptibility to a rice blast-causing fungal pathogen, Magnaporthe oryzae. These results indicate that OsSERK genes, such as OsBAK1, play versatile roles in rice growth and development.     

Activation of a Pollenin Promoter upon Nematode Infection

Three glycine-rich protein genes of Arabidopsis thaliana (Atgrp-6, Atgrp-7, and Atgrp-8) that correspond to putative genes coding for pollenins (AtolnB;2, AtolnB;3, and AtolnB;4, respectively) are expressed predominantly in the anthers and, more specifically, in the tapetum layer. Tapetal cells are responsible for nutrition of developing pollen grains and show some functional similarities to nematode feeding sites (NFS) induced in plant roots by sedentary parasitic nematodes. The aim of this study was to analyze promoter activity of the Atgrp genes in NFS. Transformed Arabidopsis plants containing a promoter-ß-glucuronidase (gus) fusion of the Atgrp-7 gene were inoculated with the root-knot nematode Meloidogyne incognita and the cyst nematode Heterodera schachtii. GUS assays were performed at different time points after infection. Histochemical analysis revealed an up-regulation of Atgrp-7-gus expression 3 days after inoculation in the feeding sites of both nematodes. Maximal Atgrp-7-gus staining levels in NFS were observed 1 week after nematode infection.