Overexpression of Arabidopsis thaliana brassinosteroid-related acyltransferase 1 gene induces brassinosteroid-deficient phenotypes in creeping bentgrass

Brassinosteroids (BRs) are naturally occurring steroidal hormones that play diverse roles in various processes during plant growth and development. Thus, genetic manipulation of endogenous BR levels might offer a way of improving the agronomic traits of crops, including plant architecture and stress tolerance. In this study, we produced transgenic creeping bentgrass (Agrostis stolonifera L.) overexpressing a BR-inactivating enzyme, Arabidopsis thaliana BR-related acyltransferase 1 (AtBAT1), which is known to catalyze the conversion of BR intermediates to inactive acylated conjugates. After putative transgenic plants were selected using herbicide resistance assay, genomic integration of the AtBAT1 gene was confirmed by genomic PCR and Southern blot analysis, and transgene expression was validated by northern blot analysis. The transgenic creeping bentgrass plants exhibited BR-deficient phenotypes, including reduced plant height with shortened internodes (i.e., semi-dwarf), reduced leaf growth rates with short, wide, and thick architecture, high chlorophyll contents, decreased numbers of vascular bundles, and large lamina joint bending angles (i.e., erect leaves). Subsequent analyses showed that the transgenic plants had significantly reduced amounts of endogenous BR intermediates, including typhasterol, 6-deoxocastasterone, and castasterone. Moreover, the AtBAT1 transgenic plants displayed drought tolerance as well as delayed senescence. Therefore, the results of the present study demonstrate that overexpression of an Arabidopsis BR-inactivating enzyme can reduce the endogenous levels of BRs in creeping bentgrass resulting in BR-deficient phenotypes, indicating that the AtBAT1 gene from a dicot plant is also functional in the monocot crop.111Ysciescopu

TOR signaling promotes accumulation of BZR1 to balance growth with carbon availability in arabidopsis

For maintenance of cellular homeostasis, the actions of growth-promoting hormones must be attenuated when nutrient and energy become limiting. The molecular mechanisms that coordinate hormone-dependent growth responses with nutrient availability remain poorly understood in plants [1, 2]. The target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates nutrient and energy signaling to regulate growth and homeostasis in both animals and plants [3-7]. Here, we show that sugar signaling through TOR controls the accumulation of the brassinosteroid (BR)-signaling transcription factor BZR1, which is essential for growth promotion by multiple hormonal and environmental signals [8-11]. Starvation, caused by shifting of light-grown Arabidopsis seedlings into darkness, as well as inhibition of TOR by inducible RNAi, led to plant growth arrest and reduced expression of BR-responsive genes. The growth arrest caused by TOR inactivation was partially recovered by BR treatment and the gain-of-function mutation bzr1-1D, which causes accumulation of active forms of BZR1 [12]. Exogenous sugar promoted BZR1 accumulation and seedling growth, but such sugar effects were largely abolished by inactivation of TOR, whereas the effect of TOR inactivation on BZR1 degradation is abolished by inhibition of autophagy and by the bzr1-1D mutation. These results indicate that cellular starvation leads sequentially to TOR inactivation, autophagy, and BZR1 degradation. Such regulation of BZR1 accumulation by glucose-TOR signaling allows carbon availability to control the growth promotion hormonal programs, ensuring supply-demand balance in plant growth

The complete mitochondrial genome of Hipparchia autonoe (Esper, 1783) (Lepidoptera: Nymphalidae): investigation of intraspecific variations on mitochondrial genome

Hipparchia autonoe (Esper, 1783) is a protected butterfly species found in Mt. Halla in South Korea. We have determined mitochondrial genome of H. autonoe collected in Mt. Halla. The circular mitogenome of H. autonoe is 15,300 bp long, which is shorter than previously sequenced mitogenome by 189 bp due to differences of tandem repeats. It includes 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNAs. The base composition was AT-biased (78.9%). Nineteen single nucleotide polymorphisms and one insertion and deletion were identified between the two individuals of H. autonoe captured in Mt. Halla, presenting enough genetic diversity of H. autonoe within population

Comparison of vascular bundles in internodes between control plant (NT) and the AtBAT1 plants.

<p>(A) Cross-sections of internodes from the second leaf of the first stoloniferous plant. (B) Measurements of the number of vascular bundles. Data represent means ± SD of three independent measurements (<i>n</i> = 20 for each measurement). Statistically significant changes compared with NT are indicated by ** at <i>P</i> < 0.001.</p

Stomatal closing analysis of the AtBAT1 plants.

<p>(A) Representative stomata observed in creeping bentgrass. The observed stomata could be classified into three types: completely open (left), partially open (middle), and completely closed (right). Bar = 5 μm. The apertures of the completely and partially open stomata are approximately 2.5 and 1.4 μm, respectively, whereas those of the completely closed stomata are not measurable. (B) The ratios of stomata closing in the absence (−) and presence (+) of ABA. The number of completely closed stomata was counted to calculate the percentages of stomatal closing. Data represent means ± SD of three independent measurements (<i>n</i> = 150 for each measurement).</p

Comparison of leaf senescence between control plant (NT) and the AtBAT1 plants.

<p>(A) Leaves after senescence treatment. The detached leaves were incubated for 2 weeks in the dark at 25°C. (B) Loss of chlorophyll content after the senescence treatment. Data represent means ± SD of three independent measurements. Statistically significant changes compared with NT are indicated by ** at <i>P</i> < 0.001.</p

Quantitative analysis of endogenous BR intermediates in creeping bentgrass.

<p>Quantitative analysis of endogenous BR intermediates in creeping bentgrass.</p

Production of transgenic creeping bentgrass plants overexpressing <i>AtBAT1</i>.

<p>(A) T-DNA region of the binary vector pCAMBIA3300 harboring the <i>AtBAT1</i> expression cassette. R, right border; L, left border; 35S^p, CaMV 35S promoter; 35S^T, 35S transcriptional terminator; NOS^T, <i>NOS</i> transcriptional terminator; <i>BAR</i>, phosphinothricin acetyltransferase gene; <i>AtBAT1</i>, <i>Arabidopsis thaliana</i> BR-related acyltransferase 1 gene. (B) Herbicide resistance assay. NT, non-transformed creeping bentgrass plant; HR, transgenic creeping bentgrass plant with an empty vector (i.e., a positive control plant with herbicide resistance). Numbers represent putative transgenic creeping bentgrass lines carrying <i>AtBAT1</i>. Herbicide resistance was determined 10 days after 0.4% BASTA^® treatment. (C) Southern blot analysis. Genomic DNA (30 μg) from each plant was digested with <i>Xba</i>I, <i>Eco</i>RI, or <i>Hind</i>III, and then hybridized with the <i>BAR</i> probe. (D) Northern blot analysis. Total RNA (15 μg) from each plant was used for hybridizations with the <i>BAR</i> or <i>AtBAT1</i> probe. Total RNA is shown as loading controls.</p

Drought tolerance of the AtBAT1 plants.

<p>(A) Drought tolerance assay. Water was withhold from 4-week-grown plants for 16 days, and images were taken before and after drought treatment. As the results of transgenic plants #4 and #5 were similar to those of #3, transgenic plant #3 is shown as a representative plant. (B) Measurements of relative water content (RWC). The leaves were harvested every 4 days after stopping irrigation, and the fresh, turgid, and dry weights were measured to calculate the RWC. Data represent mean ± SD of three independent measurements. (C) Average survival rates after drought treatment. Data represent means ± SD of three independent measurements (<i>n</i> = 60 for each measurement). Statistically significant changes compared with NT are indicated by ** at <i>P</i> < 0.001.</p