Identification, isolation and expression analysis of eight stress-related R2R3-MYB genes in tartary buckwheat (Fagopyrum tataricum).

KEY MESSAGE: Eight R2R3 - MYB genes in tartary buckwheat were identified, and their expression patterns were comprehensively analyzed, which reveals role in plant response to abiotic stresses. The proteins of the R2R3-MYB superfamily play key roles in the growth and development processes as well as defense responses in plants. However, their characteristics and functions have not been fully investigated in tartary buckwheat (Fagopyrum tataricum), a strongly abiotic resistant coarse cereal. In this article, eight tartary buckwheat R2R3-MYB genes were isolated with full-length cDNA and DNA sequences. Phylogenetic analysis of the members of the R2R3-MYB superfamily between Arabidopsis and tartary buckwheat revealed that the assumed functions of the eight tartary buckwheat R2R3-MYB proteins are divided into five Arabidopsis functional subgroups that are involved in abiotic stress. Expression analysis during abiotic stress and exogenous phytohormone treatments identified that the eight R2R3-MYB genes responded to one or more treatments. This study is the first comprehensive analysis of the R2R3-MYB gene family in tartary buckwheat under abiotic stress.

Characterization of two tartary buckwheat R2R3-MYB transcription factors and their regulation of proanthocyanidin biosynthesis.

Tartary buckwheat (Fagopyrum tataricum Gaertn.) contains high concentrations of flavonoids. The flavonoids are mainly represented by rutin, anthocyanins and proanthocyanins in tartary buckwheat. R2R3-type MYB transcription factors (TFs) play key roles in the transcriptional regulation of the flavonoid biosynthetic pathway. In this study, two TF genes, FtMYB1 and FtMYB2, were isolated from F. tataricum and characterized. The results of bioinformatic analysis indicated that the putative FtMYB1 and FtMYB2 proteins belonged to the R2R3-MYB family and displayed a high degree of similarity with TaMYB14 and AtMYB123/TT2. In vitro and in vivo evidence both showed the two proteins were located in the nucleus and exhibited transcriptional activation activities. During florescence, both FtMYB1 and FtMYB2 were more highly expressed in the flowers than any other organ. The overexpression of FtMYB1 and FtMYB2 significantly enhanced the accumulation of proanthocyanidins (PAs) and showed a strong effect on the target genes' expression in Nicotiana tabacum. The expression of dihydroflavonol-4-reductase (DFR) was upregulated to 5.6-fold higher than that of control, and the expression level was lower for flavonol synthase (FLS). To our knowledge, this is the first functional characterization of two MYB TFs from F. tataricum that control the PA pathway.

Structure and functional divergence of PIP peptide family revealed by functional studies on PIP1 and PIP2 in Arabidopsis thaliana.

PAMP-induced secreted peptide (PIP), one of the small post-translationally modified peptides (PTMPs), plays a crucial role in plant development and stress tolerance. However, little is known about functional divergence among this peptide family. Here, we studied the evolution of the PIP family in 23 plant species (10 monocotyledons and 13 dicotyledons from 7 families) and their functional divergence in Arabidopsis. A total of 128 putative PIP precursors were identified and classified into two subfamilies through phylogenetic analysis. Functional studies on AtPIP1 which represents Clade I family and AtPIP2 which represents Clade II family have shown that AtPIP2 displayed stronger immunity induction activity but weaker root growth inhibition than AtPIP1 in Arabidopsis. Transcriptome analysis of Arabidopsis seedlings treated with AtPIP1 and AtPIP2 showed that differential genes for both polypeptides were significantly enriched in similar plant defense pathways. However, Co-expression and Protein-protein interaction (PPI) analysis showed that the functions of AtprePIP2 co-expressed genes were more enriched in plant defense pathways than AtprePIP1. Molecular docking results show that AtPIP1 binds to RLK7 receptor with a more stable free energy and less binding area than AtPIP2, while hydrogen bond transfer occurs at the SGP motif position. The above results suggest that the PIP family have undergone functional divergence during evolution. Collectively, this work illustrates the relationship between PIP structure and function using Arabidopsis PIP as an example, and provides new insights into the current understanding between growth inhibition and immune responses which may be correlated but not fully coupled.

Characterization of Three Glucosyltransferase Genes in Tartary Buckwheat and Their Expression after Cold Stress.

Anthocyanins confer the red color in the hypocotyl of tartary buckwheat sprouts. Uridine diphosphate (UDP)-glucose:flavonoid 3-O-glycosyltransferase (UFGT) stabilizes anthocyanin by attaching the glucosyl moiety from UDP-glucose to the C3 hydroxyl of anthocyanin. In this study, we characterized three UFGT-like genes, designated FtUFGT1, 2, and 3 from tartary buckwheat. The results revealed that FtUFGT1, FtUFGT2, and FtUFGT3 can convert cyanidin to cyanidin 3-O-glucoside, with specific activities of 20.01 × 10(-3), 8.93 × 10(-3), and 20.24 × 10(-3) IU/mg, respectively. The active-site residues of the C-terminal domains and the N-terminal domains are important for the donor and acceptor recognition of these proteins. The expression of the three FtUFGTs paralleled the tissue-specific anthocyanin accumulation. After cold treatment, the increased content of anthocyanin was accompanied by the up-regulated expression of the three FtUFGTs. Among these three UGFT gene members, FtUFGT3 showed the highest expression level and the highest specific activity, suggesting that FtUFGT3 might be the major gene involved in anthocyanin biosynthesis. These results suggested that the FtUFGT genes, FtUFGT3 in particular, might be important candidates for anthocyanin formation in tartary buckwheat sprouts.

Proteomics identification and annotation of proteins of a cell line of Bombyx mori, BmN cells.

A cell line is an important experimental platform for biological sciences as it can basically reflect the biology of its original organism. In this study, we firstly characterized the proteome of cultured BmN cells, derived from Bombyx mori. Total 1478 proteins were identified with two or more peptides by using 1D (one-dimensional) SDS/PAGE and LTQ-Orbitrap. According to the gene ontology annotation, these proteins presented diverse pI values and molecular masses, involved in various molecular functions, including catalytic activity, binding, molecular transducer activity, motor activity, transcription regulator activity, enzyme regulator activity and antioxidant activity. Some proteins related to virus infection were also identified. These results provided us with useful information to understand the molecular mechanism of B. mori as well as antiviral immunity.

Identification of the proteome of the midgut of silkworm, Bombyx mori L., by multidimensional liquid chromatography (MDLC) LTQ-Orbitrap MS.

The midgut is the digestive apparatus of the silkworm and its proteome was studied by using nano-LC (liquid chromatography) electrospray ionization MS/MS (tandem MS). MS data were analysed by using X!Tandem searching software using different parameters and validated by using the Poisson model. A total of 90 proteins were identified and 79 proteins were described for the first time. Among the new proteins, (i) 22 proteins were closely related to the digestive function of the midgut, including 11 proteins of digestive enzymes secreted by the epithelium, eight proteins of intestine wall muscle and mechanical digestion and three proteins of peritrophic membrane that could prevent the epithelium from being mechanically rubbed; (ii) 44 proteins were involved in metabolism of substance and energy; and (iii) 11 proteins were associated with signal transduction, substance transport and cell skeleton.