Cellulose is the most abundant unique biopolymer in nature with widespread applications in bioenergy and high-value bioproducts. The large transmembrane-localized cellulose synthase (CESA) complexes (CSCs) play a pivotal role in the biosynthesis and orientation of the paracrystalline cellulose microfibrils during secondary cell wall (SCW) deposition. However, the hub CESA subunit with high potential homo/heterodimerization capacity and its functional effects on cell wall architecture, cellulose crystallinity, and saccharification efficiency remains unclear. Here, we reported the highly potent binding site containing four residues of Pro435, Trp436, Pro437, and Gly438 in the plant-conserved region (P-CR) of PalCESA4 subunit, which are involved in the CESA4-CESA8 heterodimerization. The CRISPR/Cas9-knockout mutagenesis in the predicted binding site results in physiological abnormalities, stunt growth, and deficient roots. The homozygous double substitution of W436Q and P437S and heterozygous double deletions of W436 and P437 residues potentially reduced CESA4-binding affinity resulting in normal roots, 1.5-2-fold higher plant growth and cell wall regeneration rates, 1.7-fold thinner cell wall, high hemicellulose content, 37%-67% decrease in cellulose content, high cellulose DP, 25%-37% decrease in cellulose crystallinity, and 50% increase in saccharification efficiency. The heterozygous deletion of W436 increases about 2-fold CESA4 homo/heterodimerization capacity led to the 50% decrease in plant growth and increase in cell walls thickness, cellulose content (33%), cellulose DP (20%), and CrI (8%). Our findings provide a strategy for introducing commercial CRISPR/Cas9-mediated bioengineered poplars with promising cellulose applications. We anticipate our results could create an engineering revolution in bioenergy and cellulosebased nanomaterial technologies.

microRNAs (miRNAs) are a newly discovered class of non-coding small RNAs roughly 22 nucleotides long. Increasing evidence has shown that miRNAs play multiple roles in biological processes, including development, cell proliferation, apoptosis and stress responses. The identification of miRNAs and their targets is an important need to understand their roles in the development and physiology of sweet onion (Allium cepa). In this research, several computational approaches were combined to make concise prediction of the potential miRNAs and their targets. We used previously known miRNAs from other plant species against Expressed Sequence Tags (EST) database to search for the potential miRNAs. As a result, nine potential miRNAs were identified in eight ESTs of A. cepa, belonging to eight families. We could further BLAST the mRNA database and found total 154 number of the potential targets in A. cepa based on these potential miRNAs. According to the mRNA target information provided by NCBI, most of the target mRNAs appeared to be involved in plant growth, signal transduction, development, and stress responses. Gene ontology (GO) analysis implicated these targets in 32 biological processes such as protein ubiquitination, plant hormone signalling pathways and heme biosynthesis.

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Gluten is an important protein source for human beings and a wide diversity of foods has been developed to take advantage of this protein in wheat flour. However, some individuals, suffering from Celiac Disease (CD), cannot tolerate proline and glutamine-rich gluten peptides including γ-gliadins. A life-long gluten free diet is the only known effective treatment for such patients. However, the sensitivity intension in CD patients can be controlled by RNA interference (RNAi) technology. The main aim of the present study is to develop an efficient and specific siRNA-inducing cassette, as a first and critical step for an effective targeting of mRNAs of wheat γ-gliadins. To achieve this aim, we have followed the strategy based on 200 bp in sense and antisense orientation with the ~160bp sequence of none potent siRNA-containing region from γgliadin gene as a spacer in between. The endosperm-specific γ-gliadin promoter was sub-cloned into upstream the cassette. The nucleotide alignment results validated the sequence data of γ-gliadin promoter and the direct inserts with high homology identities of 99% and 99.97%, respectively. Here, six potential and consecutively arranged-siRNA sites were predicted using computational approaches. All of these sites covered the inverted repeats region with high efficacy and performance values for triggering RNAi. Abbreviations :CD Celiac Disease, CTAB Cetyl Triethyl Ammonium Bromide, dNTPs deoxy nucleoside5´-triphosphates, dsRNA double strand RNA, hpRNA hairpin RNA, HMW High Molecular Weight, IPTG Isopropyl β-D-1-thiogalactopyranoside, kD kilo Dalton, LB Luria Bertani, LMW Low molecular weight, MFE Minimum Free Energy, mRNA messenger RNA, NCBI National Center for Biotechnological Information, PCR Polymerase Chain Reaction ,pssRNAit plant short small RNA interfering tool, PTGS Post Transcriptional Gene Silencing, RISC RNA-Induced Silencing Complex, RNAi RNA interference, siRNA small interfering RNA, UPE Unpaired Energy, `X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside,

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