RING/U-box E3 protein BIR1 interacts with and ubiquitinates barley growth repressor BROAD LEAF1.

Establishment of final leaf size in plants relies on the precise regulation of two interconnected processes, cell division and cell expansion. The barley (Hordeum vulgare) protein BROAD LEAF1 (BLF1) limits cell proliferation and leaf growth in the width direction. However, how the levels of this potent repressor of leaf growth are controlled remains unclear. Here we used a yeast two-hybrid screen to identify the BLF1-INTERACTING RING/U-BOX 1 (BIR1) E3 ubiquitin ligase that interacts with BLF1 and confirmed the interaction of the two proteins in planta. Inhibiting the proteasome caused overaccumulation of a BLF1-eGFP fusion protein when co-expressed with BIR1, and an in vivo ubiquitination assay in bacteria confirmed that BIR1 can mediate ubiquitination of BLF1 protein. Consistent with regulation of endogenous BLF1 in barley by proteasomal degradation, inhibition of the proteasome in BLF1-vYFP-expressing barley plants caused an accumulation of the BLF1 protein. The BIR1 protein co-localized with BLF1 in nuclei and appeared to reduce BLF1 protein levels. Analysis of bir1-1 knock-out mutants suggested the involvement of BIR1 in leaf growth control, although mainly on leaf length. Together, our results suggest that proteasomal degradation, in part mediated by BIR1, helps fine-tune BLF1 protein levels in barley.

The gain-of-function mutation blf13 in the barley orthologue of the rice growth regulator NARROW LEAF1 is associated with increased leaf width.

Canopy architecture in cereals plays an important role in determining yield. Leaf width represents one key aspect of this canopy architecture. However, our understanding of leaf width control in cereals remains incomplete. Classical mutagenesis studies in barely identified multiple morphological mutants, including those with differing leaf widths. Of these, we characterized the broad leaf13 (blf13) mutant in detail. Mutant plants form wider leaves due to increased post-initiation growth and cell proliferation. The mutant phenotype perfectly co-segregated with a missense mutation in the HvHNT1 gene which affected a highly conserved region of the encoded protein, orthologous to the rice NARROW LEAF1 (NAL1) protein. Causality of this mutation for the blf13 phenotype is further supported by correlative transcriptomic analyses and protein-protein interaction studies showing that the mutant HvNHT1 protein interacts more strongly with a known interactor than wild-type HvHNT1. The mutant HvHNT1 protein also showed stronger homodimerization compared with wild-type HvHNT1, and homology modelling suggested an additional interaction site between HvHNT1 monomers due to the blf13 mutation. Thus, the blf13 mutation parallels known gain-of-function NAL1 alleles in rice that increase leaf width and grain yield, suggesting that the blf13 mutation may have a similar agronomic potential in barley.

Efficacy of Diclofenac in Pain Reduction after Topography-Guided Transepithelial Surface Ablation.

PURPOSE: To report the efficacy of postoperative diclofenac eye drops for pain reduction in patients undergoing topography-guided transepithelial surface ablation. METHODS: Retrospective consecutive case series of patients undergoing topography-guided transepithelial surface ablation for refractive myopia treatment using a 1 KHz excimer laser. Patients were divided into three groups. Group 1 did not receive any diclofenac drops, group 2 received one diclofenac drop postoperatively (day 1), and group 3 received one drop of diclofenac postoperatively (day 1) and on the day after treatment (day 2). Postoperative pain was self-assessed by patients per eye daily for the first 4 consecutive days (days 1 to 4) after the treatment using the visual analogue scale (VAS). We compared VAS with respect to the use of additional oral or topical treatment as well as VAS with regards to the total amount of ablated tissue (< 50 µm, 50 - 100 µm, ≥ 100 µm) among the groups. RESULTS: We enrolled 163 eyes of 163 patients (55.0% female), with a mean age of 31.3 years (SD ± 6.6; range 21 - 68). We excluded 16 patients who applied other additional analgesics. Group 1 comprised 35 eyes (21%), group 2 had 21 eyes (13%), and group 3 consisted of 107 eyes (66%). Median pain score (VAS) was 5 (range 0, 10) in group 1, which was higher than in groups 2 (median 1, range 0 to 7) and 3 (median 1.5, range 0 to 7) on the day of surgery (p < 0.0001). Percentage of patients using an additional oral NSAID on days 1 and 2 was significantly higher in group 1 (69/83%) when compared to groups 2 (24/43%) or 3 (31/49%) (p < 0.001 day 1, p = 0.001 day 2). No correlation was found between pain sensation and maximum ablation depth (Spearman correlation p > 0.05). CONCLUSION: The instillation of one drop of diclofenac after topography-guided transepithelial surface ablation reduced subjective pain sensation according to VAS and decreased the need for additional topical anesthetic drops or oral NSAID.

Semi-dwarfism and lodging tolerance in tef (Eragrostis tef) is linked to a mutation in the α-Tubulin 1 gene.

Genetic improvement of native crops is a new and promising strategy to combat hunger in the developing world. Tef is the major staple food crop for approximately 50 million people in Ethiopia. As an indigenous cereal, it is well adapted to diverse climatic and soil conditions; however, its productivity is extremely low mainly due to susceptibility to lodging. Tef has a tall and weak stem, liable to lodge (or fall over), which is aggravated by wind, rain, or application of nitrogen fertilizer. To circumvent this problem, the first semi-dwarf lodging-tolerant tef line, called kegne, was developed from an ethyl methanesulphonate (EMS)-mutagenized population. The response of kegne to microtubule-depolymerizing and -stabilizing drugs, as well as subsequent gene sequencing and segregation analysis, suggests that a defect in the α-Tubulin gene is functionally and genetically tightly linked to the kegne phenotype. In diploid species such as rice, homozygous mutations in α-Tubulin genes result in extreme dwarfism and weak stems. In the allotetraploid tef, only one homeologue is mutated, and the presence of the second intact α-Tubulin gene copy confers the agriculturally beneficial semi-dwarf and lodging-tolerant phenotype. Introgression of kegne into locally adapted and popular tef cultivars in Ethiopia will increase the lodging tolerance in the tef germplasm and, as a result, will improve the productivity of this valuable crop.

The INDETERMINATE DOMAIN Protein BROAD LEAF1 Limits Barley Leaf Width by Restricting Lateral Proliferation.

Variation in the size, shape, and positioning of leaves as the major photosynthetic organs strongly impacts crop yield, and optimizing these aspects is a central aim of cereal breeding [1, 2]. Leaf growth in grasses is driven by cell proliferation and cell expansion in a basal growth zone [3]. Although several factors influencing final leaf size and shape have been identified from rice and maize [4-14], what limits grass leaf growth in the longitudinal or transverse directions during leaf development remains poorly understood. To identify factors involved in this process, we characterized the barley mutant broad leaf1 (blf1). Mutants form wider but slightly shorter leaves due to changes in the numbers of longitudinal cell files and of cells along the leaf length. These differences arise during primordia outgrowth because of more cell divisions in the width direction increasing the number of cell files. Positional cloning, analysis of independent alleles, and transgenic complementation confirm that BLF1 encodes a presumed transcriptional regulator of the INDETERMINATE DOMAIN family. In contrast to loss-of-function mutants, moderate overexpression of BLF1 decreases leaf width below wild-type levels. A functional BLF1-vYFP fusion protein expressed from the endogenous promoter shows a dynamic expression pattern in the shoot apical meristem and young leaf primordia. Thus, we propose that the BLF1 gene regulates barley leaf size by restricting cell proliferation in the leaf-width direction. Given the agronomic importance of canopy traits in cereals, identifying functionally different BLF1 alleles promises to allow for the generation of optimized cereal ideotypes.