Manipulating a Single Transcription Factor, Ant1, Promotes Anthocyanin Accumulation in Barley Grains.

Barley has abundant anthocyanin-rich accessions, which renders it an ideal model to investigate the regulatory mechanism of anthocyanin biosynthesis. This study functionally characterized two transcription factors: Ant1 and Ant2. Sequence alignment showed that the coding sequences of Ant1 and Ant2 are conserved among 11 colored hulless barley and noncolored barley varieties. The expression profiles of Ant1 and Ant2 were divergent between species, and significantly higher expression was found in two colored Qingke accessions. The co-expression of Ant1 and Ant2 resulted in purple pigmentation in transient transformation systems via the promotion of the transcription of four structural genes. Ant1 interacted with Ant2, and overexpression of Ant1 activated the transcription of Ant2. Moreover, overexpression of Ant1 led to anthocyanin accumulation in the pericarp and aleurone layer of transgenic barley grains. Overall, our results suggest that anthocyanin-enriched barley grains can be produced by manipulating Ant1 expression.

Identification of regulatory factors promoting embryogenic callus formation in barley through transcriptome analysis.

BACKGROUND: Barley is known to be recalcitrant to tissue culture, which hinders genetic transformation and its biotechnological application. To date, the ideal explant for transformation remains limited to immature embryos; the mechanism underlying embryonic callus formation is elusive. RESULTS: This study aimed to uncover the different transcription regulation pathways between calli formed from immature (IME) and mature (ME) embryos through transcriptome sequencing. We showed that incubation of embryos in an auxin-rich medium caused dramatic changes in gene expression profiles within 48 h. Overall, 9330 and 11,318 differentially expressed genes (DEGs) were found in the IME and ME systems, respectively. 3880 DEGs were found to be specific to IME_0h/IME_48h, and protein phosphorylation, regulation of transcription, and oxidative-reduction processes were the most common gene ontology categories of this group. Twenty-three IAA, fourteen ARF, eight SAUR, three YUC, and four PIN genes were found to be differentially expressed during callus formation. The effect of callus-inducing medium (CIM) on IAA genes was broader in the IME system than in the ME system, indicating that auxin response participates in regulating cell reprogramming during callus formation. BBM, LEC1, and PLT2 exhibited a significant increase in expression levels in the IME system but were not activated in the ME system. WUS showed a more substantial growth trend in the IME system than in the ME system, suggesting that these embryonic, shoot, and root meristem genes play crucial roles in determining the acquisition of competency. Moreover, epigenetic regulators, including SUVH3A, SUVH2A, and HDA19B/703, exhibited differential expression patterns between the two induction systems, indicating that epigenetic reprogramming might contribute to gene expression activation/suppression in this process. Furthermore, we examined the effect of ectopic expression of HvBBM and HvWUS on Agrobacterium-mediated barley transformation. The transformation efficiency in the group expressing the PLTPpro:HvBBM + Axig1pro:HvWUS construct was increased by three times that in the control (empty vector) because of enhanced plant regeneration capacity. CONCLUSIONS: We identified some regulatory factors that might contribute to the differential responses of the two explants to callus induction and provide a promising strategy to improve transformation efficiency in barley.

Functional dissection of HGGT and HPT in barley vitamin E biosynthesis via CRISPR/Cas9-enabled genome editing.

BACKGROUND AND AIMS: Vitamin E (tocochromanol) is a lipid-soluble antioxidant and an essential nutrient for human health. Among cereal crops, barley (Hordeum vulgare) contains a high level of vitamin E, which includes both tocopherols and tocotrienols. Although the vitamin E biosynthetic pathway has been characterized in dicots, such as Arabidopsis, which only accumulate tocopherols, knowledge regarding vitamin E biosynthesis in monocots is limited because of the lack of functional mutants. This study aimed to obtain gene knockout mutants to elucidate the genetic control of vitamin E composition in barley. METHODS: Targeted knockout mutations of HvHPT and HvHGGT in barley were created with CRISPR/Cas9-enabled genome editing. High-performance liquid chromatography (HPLC) was performed to analyse the content of tocochromanol isomers in transgene-free homozygous Hvhpt and Hvhggt mutants. KEY RESULTS: Mutagenesis efficiency among T0 regenerated plantlets was 50-65 % as a result of two simultaneously expressed guide RNAs targeting each gene; most of the mutations were stably inherited by the next generation. The transgene-free homozygous mutants of Hvhpt and Hvhggt exhibited decreased grain size and weight, and the HvHGGT mutation led to a shrunken phenotype and significantly lower total starch content in grains. HPLC analysis revealed that targeted mutation of HvHPT significantly reduced the content of both tocopherols and tocotrienols, whereas mutations in HvHGGT completely blocked tocotrienol biosynthesis in barley grains. Transient overexpression of an HvHPT homologue in tobacco leaves significantly increased the production of γ- and δ-tocopherols, which may partly explain why targeted mutation of HvHPT in barley grains did not eliminate tocopherol production. CONCLUSIONS: Our results functionally validated that HvHGGT is the only committed gene for the production of tocotrienols, whereas HvHPT is partly responsible for tocopherol biosynthesis in barley.

The barley miR393 has multiple roles in regulation of seedling growth, stomatal density, and drought stress tolerance.

microRNA393 (miR393) and its target module have been implicated as comprising a conserved mechanism to regulate developmental processes and plant growth in response to environmental signals through the auxin signaling pathway. Our previous work identified miR393 and its two targets in barley. In this study, we further investigated the expression pattern of miR393 and its biological functions in seedling growth and drought tolerance. We showed that the miR393 overexpressing line (OE) exhibited increased stomatal density with decreased guard cell length, while the miR393 knockdown line (MIM) displayed the opposite phenotype, which might be due to the effects of miR393 on AUXIN RESPONSE FACTOR5 (ARF5) and three stomatal development-related genes, such as EPIDERMAL PATTERNING FACTOR1 (EPF1), SPEECHLESS (SPCH), and MUTE. In addition, the MIM line conferred enhanced drought tolerance, with alleviated leaf chlorosis and lipid peroxidation after 22 days drought treatment. In contrast, the OE line was more sensitive to drought stress and accumulated more malondialdehyde and hydrogen peroxide than the wild type. Furthermore, polyethylene glycol (PEG) treatment-induced abscisic acid (ABA) accumulation in leaves was suppressed in the OE line, indicating that miR393 might regulate drought stress response and tolerance through its interaction with ABA biosynthesis. Overall, these data suggest that miR393 might be a potential target for manipulation of stomatal density and improvement of drought tolerance in barley.

Wavefront correction by a low-cost deformable mirror group in a small-aperture-beam fiber laser.

A low-cost deformable mirror (DM) group for wavefront correction in a small-aperture-beam fiber laser is reported in this paper. The DM group consists of nine single-actuator DMs and could act as a "virtual" 9-actuator DM arranged in a 3×3 array. The equivalent distance between two adjacent actuators could reach as small as 3 mm, which can lead to a relatively high lateral resolution. Reflection mirrors in the DM group can be individually polished and high-damage-threshold coated. The unique manufacture flexibility will ensure the particularly low cost of the DM group. In this paper, a detailed configuration of the DM group and its application in small-aperture-beam fiber lasers are presented. Theoretic simulations and experimental results successfully demonstrate its capability in wavefront aberration correction for a small-aperture-beam fiber laser.

High-precision system identification method for a deformable mirror in wavefront control.

Based on a mathematic model, the relation between the accuracy of the influence matrix and the performance of the wavefront correction is established. Based on the least squares method, a two-step system identification is proposed to improve the accuracy of the influence matrix, where the measurement noise can be suppressed and the nonlinearity of the deformable mirror can be compensated. The validity of the two-step system identification method is tested in the experiment, where improvements in wavefront correction precision as well as closed-loop control efficiency were observed.