Light-Induced TaHY5-7A and TaBBX-3B Physically Interact to Promote PURPLE PERICARP-MYB 1 Expression in Purple-Grained Wheat.

Purple-grained wheat (Triticum aestivum L.) is an important germplasm source in crop breeding. Anthocyanin biosynthesis in the pericarps of purple-grained wheat is largely light-dependent; however, the regulatory mechanisms underlying light-induced anthocyanin accumulation in the wheat pericarp remain unknown. Here we determined that anthocyanins rapidly accumulate in the pericarps of the purple-grained wheat cultivar Heixiaomai 76 (H76) at 16 days after pollination under light treatment. Using transcriptome sequencing, differential gene expression analysis, and phylogenetic analysis, we identified two key genes involved in light signaling in wheat: ELONGATED HYPOCOTYL 5-7A (TaHY5-7A) and B-BOX-3B (TaBBX-3B). TaHY5-7A and TaBBX-3B were highly expressed in purple-grained wheat pericarps. The heterologous expression of TaHY5-7A partially restored the phenotype of the Arabidopsis (Arabidopsis thaliana) hy5 mutant, resulting in increased anthocyanin accumulation and a shortened hypocotyl. The heterologous expression of TaBBX-3B in wild-type Arabidopsis had similar effects. TaHY5-7A and TaBBX-3B were nucleus-localized, consistent with a function in transcription regulation. However, TaHY5-7A, which lacks a transactivation domain, was not sufficient to activate the expression of PURPLE PERICARP-MYB 1 (TaPpm1), the key anthocyanin biosynthesis regulator in purple pericarps of wheat. TaHY5-7A physically interacted with TaBBX-3B in yeast two-hybrid and bimolecular fluorescence complementation assays. Additionally, TaHY5-7A, together with TaBBX-3B, greatly enhanced the promoter activity of TaPpm1 in a dual luciferase assay. Overall, our results suggest that TaHY5-7A and TaBBX-3B collaboratively activate TaPpm1 expression to promote light-induced anthocyanin biosynthesis in purple-pericarp wheat.

The chloroplast genome of Amygdalus L. (Rosaceae) reveals the phylogenetic relationship and divergence time.

BACKGROUND: Limited access to genetic information has greatly hindered our understanding of the molecular evolution, phylogeny, and differentiation time of subg. Amygdalus. This study reported complete chloroplast (cp) genome sequences of subg. Amygdalus, which further enriched the available valuable resources of complete cp genomes of higher plants and deepened our understanding of the divergence time and phylogenetic relationships of subg. Amygdalus. RESULTS: The results showed that subg. Amygdalus species exhibited a tetrad structure with sizes ranging from 157,736 bp (P. kansuensis) to 158,971 bp (P. davidiana), a pair of inverted repeat regions (IRa/IRb) that ranged from 26,137-26,467 bp, a large single-copy region that ranged from 85,757-86,608 bp, and a small single-copy region that ranged from 19,020-19,133 bp. The average GC content of the complete cp genomes in the 12 species was 36.80%. We found that the structure of the subg. Amygdalus complete cp genomes was highly conserved, and the 12 subg. Amygdalus species had an rps19 pseudogene. There was not rearrangement of the complete cp genome in the 12 subg. Amygdalus species. All 12 subg. Amygdalus species clustered into one clade based on both Bayesian inference and maximum likelihood. The divergence time analyses based on the complete cp genome sequences showed that subg. Amygdalus species diverged approximately 15.65 Mya. CONCLUSION: Our results provide data on the genomic structure of subg. Amygdalus and elucidates their phylogenetic relationships and divergence time.

Three endoplasmic reticulum-associated fatty acyl-coenzyme a reductases were involved in the production of primary alcohols in hexaploid wheat (Triticum aestivum L.).

BACKGROUND: The cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between the plant and the environment. The cuticle is composed of cutin and wax. Cuticular wax plays an important role in the survival of plants by serving as the interface between plants and their biotic and abiotic environments, especially restricting nonstomatal water loss. Leaf cuticular waxes of hexaploid wheat at the seedling stage mainly consist of primary alcohols, aldehydes, fatty acids, alkane and esters. Primary alcohols account for more than 80% of the total wax load. Therefore, we cloned several genes encoding fatty acyl-coenzyme A reductases from wheat and analyzed their function in yeast and plants. We propose the potential use of these genes in wheat genetic breeding. RESULTS: We reported the cloning and characterization of three TaFARs, namely TaFAR6, TaFAR7 and TaFAR8, encoding fatty acyl-coenzyme A reductases (FAR) in wheat leaf cuticle. Expression analysis revealed that TaFAR6, TaFAR7 and TaFAR8 were expressed at the higher levels in the seedling leaf blades, and were expressed moderately or weakly in stamen, glumes, peduncle, flag leaf blade, sheath, spike, and pistil. The heterologous expression of three TaFARs in yeast (Saccharomyces cerevisiae) led to the production of C24:0 and C26:0 primary alcohols. Transgenic expression of the three TaFARs in tomato (Solanum lycopersicum) and rice (Oryza sativa) led to increased accumulation of C24:0-C30:0 primary alcohols. Transient expression of GFP protein-tagged TaFARs revealed that the three TaFAR proteins were localized to the endoplasmic reticulum (ER), the site of wax biosynthesis. The three TaFAR genes were transcriptionally induced by drought, cold, heat, powdery mildew (Blumeria graminis) infection, abscisic acid (ABA) and methyl jasmonate (MeJa) treatments. CONCLUSIONS: These results indicated that wheat TaFAR6, TaFAR7 and TaFAR8 are involved in biosynthesis of very-long-chain primary alcohols in hexaploid wheat and in response to multiple environmental stresses.

Three Fatty Acyl-Coenzyme A Reductases, BdFAR1, BdFAR2 and BdFAR3, are Involved in Cuticular Wax Primary Alcohol Biosynthesis in Brachypodium distachyon.

Plant cuticular wax is a heterogeneous mixture of very long chain fatty acids (VLCFAs) and their derivatives. Primary alcohols are the dominant wax components throughout leaf development of Brachypodium distachyon (Brachypodium). However, the genes involved in primary alcohol biosynthesis have not been investigated and their exact biological function remains unclear in Brachypodium to date. Here, we monitored the leaf wax profile and crystal morphology during Brachypodium leaf morphogenesis, and isolated three Brachypodium fatty acyl-CoA reductase (FAR) genes, named BdFAR1, BdFAR2 and BdFAR3, then analyzed their biochemical activities, substrate specificities, expression patterns, subcellular localization and stress induction. Transgenic expression of BdFAR genes in yeast (Saccharomyces cerevisiae), tomato (Solanum lycopersicum), Arabidopsis (Arabidopsis thaliana) and Brachypodium increased the production of primary alcohols. The three BdFAR genes were preferentially expressed in Brachypodium aerial tissues, consistent with known sites of wax primary alcohol deposition, and localized in the endoplasmic reticulum (ER) in Arabidopsis protoplasts. Finally, expression of the BdFAR genes was induced by drought, cold and ABA treatments, and drought stress significantly increased cuticular wax accumulation in Brachypodium. Taken together, these results indicate that the three BdFAR genes encode active FARs involved in the biosynthesis of Brachypodium wax primary alcohols and respond to abiotic stresses.

De novo assembly and transcriptome analysis of two contrary tillering mutants to learn the mechanisms of tillers outgrowth in switchgrass (Panicum virgatum L.).

Tillering is an important trait in monocotyledon plants. The switchgrass (Panicum virgatum), studied usually as a source of biomass for energy production, can produce hundreds of tillers in its lifetime. Studying the tillering of switchgrass also provides information for other monocot crops. High-tillering and low-tillering mutants were produced by ethyl methanesulfonate mutagenesis. Alteration of tillering ability resulted from different tiller buds outgrowth in the two mutants. We sequenced the tiller buds transcriptomes of high-tillering and low-tillering plants using next-generation sequencing technology, and generated 34 G data in total. In the de novo assembly results, 133,828 unigenes were detected with an average length of 1,238 bp, and 5,290 unigenes were differentially expressed between the two mutants, including 3,225 up-regulated genes and 2,065 down-regulated genes. Differentially expressed gene analysis with functional annotations was performed to identify candidate genes involved in tiller bud outgrowth processes using Gene Ontology classification, Cluster of Orthologous Groups of proteins, and Kyoto Encyclopedia of Genes and Genomes pathway analysis. This is the first study to explore the tillering transcriptome in two types of tillering mutants by de novo sequencing.

Developmental Changes in Composition and Morphology of Cuticular Waxes on Leaves and Spikes of Glossy and Glaucous Wheat (Triticum aestivum L.).

The glossy varieties (A14 and Jing 2001) and glaucous varieties (Fanmai 5 and Shanken 99) of wheat (Triticum aestivum L.) were selected for evaluation of developmental changes in the composition and morphology of cuticular waxes on leaves and spikes. The results provide us with two different wax development patterns between leaf and spike. The general accumulation trend of the total wax load on leaf and spike surfaces is first to increase and then decrease during the development growth period, but these changes were caused by different compound classes between leaf and spike. Developmental changes of leaf waxes were mainly the result of variations in composition of alcohols and alkanes. In addition, diketones were the third important contributor to the leaf wax changes in the glaucous varieties. Alkanes and diketones were the two major compound classes that caused the developmental changes of spike waxes. For leaf waxes, ß- and OH-ß-diketones were first detected in flag leaves from 200-day-old plants, and the amounts of ß- and OH-ß-diketones were significantly higher in glaucous varieties compared with glossy varieties. In spike waxes, ß-diketone existed in all varieties, but OH-ß-diketone was detectable only in the glaucous varieties. Unexpectedly, the glaucous variety Fanmai 5 yielded large amounts of OH-ß-diketone. There was a significant shift in the chain length distribution of alkanes between early stage leaf and flag leaf. Unlike C28 alcohol being the dominant chain length in leaf waxes, the dominant alcohol chain length of spikes was C24 or C26 depending on varieties. Epicuticular wax crystals on wheat leaf and glume were comprised of platelets and tubules, and the crystal morphology changed constantly throughout plant growth, especially the abaxial leaf crystals. Moreover, our results suggested that platelets and tubules on glume surfaces could be formed rapidly within a few days.

FAR5, a fatty acyl-coenzyme A reductase, is involved in primary alcohol biosynthesis of the leaf blade cuticular wax in wheat (Triticum aestivum L.).

A waxy cuticle that serves as a protective barrier against non-stomatal water loss and environmental damage coats the aerial surfaces of land plants. It comprises a cutin polymer matrix and waxes. Cuticular waxes are complex mixtures of very long chain fatty acids (VLCFAs) and their derivatives. Results show that primary alcohols are the major components of bread wheat (Triticum aestivum L.) leaf blade cuticular waxes. Here, the characterization of TaFAR5 from wheat cv Xinong 2718, which is allelic to TAA1b, an anther-specific gene, is reported. Evidence is presented for a new function for TaFAR5 in the biosynthesis of primary alcohols of leaf blade cuticular wax in wheat. Expression of TaFAR5 cDNA in yeast (Saccharomyces cerevisiae) led to production of C22:0 primary alcohol. The transgenic expression of TaFAR5 in tomato (Solanum lycopersicum) cv MicroTom leaves resulted in the accumulation of C26:0, C28:0, and C30:0 primary alcohols. TaFAR5 encodes an alcohol-forming fatty acyl-coenzyme A reductase (FAR). Expression analysis revealed that TaFAR5 was expressed at high levels in the leaf blades, anthers, pistils, and seeds. Fully functional green fluorescent protein-tagged TaFAR5 protein was localized to the endoplasmic reticulum (ER), the site of primary alcohol biosynthesis. SDS-PAGE analysis indicated that the TaFAR5 protein possessed a molecular mass of 58.4kDa, and it was also shown that TaFAR5 transcript levels were regulated in response to drought, cold, and abscisic acid (ABA). Overall, these data suggest that TaFAR5 plays an important role in the synthesis of primary alcohols in wheat leaf blade.