The miRNAome of \u3cem\u3eCatharanthus roseus\u3c/em\u3e: Identification, Expression Analysis, and Potential Roles of MicroRNAs in Regulation of Terpenoid Indole Alkaloid Biosynthesis

MicroRNAs (miRNAs) regulate numerous crucial biological processes in plants. However, information is limited on their involvement in the biosynthesis of specialized metabolites in plants, including Catharanthus roseus that produces a number of pharmaceutically valuable, bioactive terpenoid indole alkaloids (TIAs). Using small RNA-sequencing, we identified 181 conserved and 173 novel miRNAs (cro-miRNAs) in C. roseus seedlings. Genome-wide expression analysis revealed that a set of cro-miRNAs are differentially regulated in response to methyl jasmonate (MeJA). In silico target prediction identified 519 potential cro-miRNA targets that include several auxin response factors (ARFs). The presence of cleaved transcripts of miRNA-targeted ARFs in C. roseus cells was confirmed by Poly(A) Polymerase-Mediated Rapid Amplification of cDNA Ends (PPM-RACE). We showed that auxin (indole acetic acid, IAA) repressed the expression of key TIA pathway genes in C. roseus seedlings. Moreover, we demonstrated that a miRNA-regulated ARF, CrARF16, binds to the promoters of key TIA pathway genes and repress their expression. The C. roseus miRNAome reported here provides a comprehensive account of the cro-miRNA populations, as well as their abundance and expression profiles in response to MeJA. In addition, our findings underscore the importance of miRNAs in posttranscriptional control of the biosynthesis of specialized metabolites

RNA-Sequencing Reveals Global Transcriptomic Changes in \u3cem\u3eNicotiana tabacum\u3c/em\u3e Responding to Topping and Treatment of Axillary-Shoot Control Chemicals

Removal of terminal buds (topping) and control of the formation of axillary shoots (suckers) are common agronomic practices that significantly impact the yield and quality of various crop plants. Application of chemicals (suckercides) to plants following topping is an effective method for sucker control. However, our current knowledge of the influence of topping, and subsequent suckercide applications, to gene expression is limited. We analyzed the differential gene expression using RNA-sequencing in tobacco (Nicotiana tabacum) that are topped, or treated after topping by two different suckercides, the contact-localized-systemic, Flupro® (FP), and contact, Off-Shoot-T®. Among the differentially expressed genes (DEGs), 179 were identified as common to all three conditions. DEGs, largely related to wounding, phytohormone metabolism and secondary metabolite biosynthesis, exhibited significant upregulation following topping, and downregulation after suckercide treatments. DEGs related to photosynthetic processes were repressed following topping and suckercide treatments. Moreover, topping and FP-treatment affect the expression of auxin and cytokinin signaling pathway genes that are possibly involved in axillary shoot formation. Our results provide insights into the global change of plant gene expression in response to topping and suckercide treatments. The regulatory elements of topping-inducible genes are potentially useful for the development of a chemical-free sucker control system

Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae

Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K+) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K+ channel proteins consisting of 82 amino acids. The KcvATCV-1 protein has similarities to the family of two transmembrane domain K+ channel proteins; it consists of two transmembrane -helixes with a pore region in the middle, making it an ideal model for studying K+ channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from KcvATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K+ channels

Identification of a Chlorovirus PBCV-1 Protein Involved in Degrading the Host Cell Wall during Virus Infection

Chloroviruses are unusual among viruses infecting eukaryotic organisms in that they must, like bacteriophages, penetrate a rigid cell wall to initiate infection. Chlorovirus PBCV-1 infects its host, Chlorella variabilis NC64A by specifically binding to and degrading the cell wall of the host at the point of contact by a virus-packaged enzyme(s). However, PBCV-1 does not use any of the five previously characterized virus-encoded polysaccharide degrading enzymes to digest the Chlorella host cell wall during virus entry because none of the enzymes are packaged in the virion. A search for another PBCV-1-encoded and virion-associated protein identified protein A561L. The fourth domain of A561L is a 242 amino acid C-terminal domain, named A561LD4, with cell wall degrading activity. An A561LD4 homolog was present in all 52 genomically sequenced chloroviruses, infecting four different algal hosts. A561LD4 degraded the cell walls of all four chlorovirus hosts, as well as several non-host Chlorella spp. Thus, A561LD4 was not cell-type specific. Finally, we discovered that exposure of highly purified PBCV-1 virions to A561LD4 increased the specific infectivity of PBCV-1 from about 25–30% of the particles forming plaques to almost 50%. We attribute this increase to removal of residual host receptor that attached to newly replicated viruses in the cell lysates

Screening of superior fiber-quality-traits among wild accessions of

• The search for alternative fiber resources is receiving worldwide attention for the potential role of these fibers in climate and forest restoration. Among bamboos, Bambusa balcooa is generally preferred for industrial use due to its rapid growth, excellent flexibility and tensile strength. The aim of this study is to establish a non-invasive and efficient method of screening superior fiber quality from the wild gene pool of B. balcooa. • Anatomical features of B. balcooa internodes were revealed by light microscopy. Confocal laser scanning microscopy (CLSM) was used to study fiber developmental stages in emerging branches using the intrinsic fluorescence properties of lignin and chlorophyll. Physical properties of B. balcooa fibers from different gene pools were studied, and biochemical estimations of lignin and cellulose contents were performed. CLSM-based semi-quantitative in situ estimations of cellulose and lignin contents in fiber bundles were performed using orthochromatic Congo red (CR) and acridine orange (AO) labeling, respectively. • Fibers isolated from representative accessions of locations 3 and 4 showed the highest cellulose contents and superior physical properties. The results obtained from biochemical analyses of cellulose and lignin contents of fiber bundles among the accessions correlate significantly with CR and AO fluorescence values using non-invasive methods. A CLSM-based study demonstrated the possibility of differentiating stages of fiber development with autofluorescent markers of lignin and lignin precursors that would help to define stages of fiber development and maturation in bamboo effectively, which is a prerequisite for understanding the mechanism of fiber development at the molecular level in bamboo. • A CLSM-based cellulose and lignin assay is a non-invasive method, exploited for the first time for genetic diversity screening in a wild gene pool. This protocol would also be useful for rapid resource screening for various commercial purposes

Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae

Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K+) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K+ channel proteins consisting of 82 amino acids. The KcvATCV-1 protein has similarities to the family of two transmembrane domain K+ channel proteins; it consists of two transmembrane -helixes with a pore region in the middle, making it an ideal model for studying K+ channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from KcvATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K+ channels

Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae

Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K channel proteins consisting of 82 amino acids. The Kcv protein has similarities to the family of two transmembrane domain K channel proteins; it consists of two transmembrane α-helixes with a pore region in the middle, making it an ideal model for studying K channels. To assess their genetic diversity, genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from Kcv by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K channels