Virus-Mediated Transient Expression Techniques Enable Functional Genomics Studies and Modulations of Betalain Biosynthesis and Plant Height in Quinoa

スーパー作物キヌアにおける遺伝子機能の解析技術を開発 --優れた環境適応性や栄養特性の謎を解き、作物開発を加速化--. 京都大学プレスリリース. 2021-03-19.Quinoa (Chenopodium quinoa), native to the Andean region of South America, has been recognized as a potentially important crop in terms of global food and nutrition security since it can thrive in harsh environments and has an excellent nutritional profile. Even though challenges of analyzing the complex and heterogeneous allotetraploid genome of quinoa have recently been overcome, with the whole genome-sequencing of quinoa and the creation of genotyped inbred lines, the lack of technology to analyze gene function in planta is a major limiting factor in quinoa research. Here, we demonstrate that two virus-mediated transient expression techniques, virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX), can be used in quinoa. We show that apple latent spherical virus (ALSV) can induce gene silencing of quinoa phytoene desaturase (CqPDS1) in a broad range of quinoa inbred lines derived from the northern and southern highland and lowland sub-populations. In addition, we show that ALSV can be used as a VOX vector in roots. Our data also indicate that silencing a quinoa 3, 4-dihydroxyphenylalanine 4, 5-dioxygenase gene (CqDODA1) or a cytochrome P450 enzyme gene (CqCYP76AD1) inhibits betalain production and that knockdown of a reduced-height gene homolog (CqRHT1) causes an overgrowth phenotype in quinoa. Moreover, we show that ALSV can be transmitted to the progeny of quinoa plants. Thus, our findings enable functional genomics in quinoa, ushering in a new era of quinoa research

Virus-induced down-regulation of <i>GmERA1A</i> and <i>GmERA1B</i> genes enhances the stomatal response to abscisic acid and drought resistance in soybean

<div><p>Drought is a major threat to global soybean production. The limited transformation potential and polyploid nature of soybean have hindered functional analysis of soybean genes. Previous research has implicated farnesylation in the plant’s response to abscisic acid (ABA) and drought tolerance. We therefore used virus-induced gene silencing (VIGS) to evaluate farnesyltransferase genes, <i>GmERA1A</i> and <i>GmERA1B</i> (<i>Glycine max Enhanced Response to ABA1-A</i> and <i>-B</i>), as potential targets for increasing drought resistance in soybean. <i>Apple latent spherical virus</i> (ALSV)-mediated <i>GmERA1</i>-down-regulated soybean leaves displayed an enhanced stomatal response to ABA and reduced water loss and wilting under dehydration conditions, suggesting that GmERA1A and GmERA1B negatively regulate ABA signaling in soybean guard cells. The findings provide evidence that the ALSV-VIGS system, which bypasses the need to generate transgenic plants, is a useful tool for analyzing gene function using only a single down-regulated leaf. Thus, the ALSV-VIGS system could constitute part of a next-generation molecular breeding pipeline to accelerate drought resistance breeding in soybean.</p></div

Evaluation of drought resistance in ALSV-GmERA1-infected soybean plants.

<p>Watering was withheld from plants at the V6 growth stage for 3 days. The drought status of the fourth to sixth trifoliate leaves was scored from 0 to 4 as follows: 0, no change; 1, wilting; 2, rolling; and 3, severe wilting. (A) Representative photographs of leaves with various scores. The leaves were scored at several time points after water withholding. (B) Leaf rolling score before rehydration (72–75 h after water withholding). The trial was repeated independently six times. (C) Plants were re-watered at 3 days after water withholding. Representative plants at one day after rehydration are shown.</p

<i>GmERA1</i>-down-regulated leaves show reduced water loss rates and stomatal conductance.

<p>(A) Rates of water loss in the fourth or fifth trifoliate leaves of soybean plants inoculated with ALSV vectors. Each data point represents the mean of duplicate measurements (<i>n</i> = 3 for ALSV-VC and <i>n</i> = 6 for ALSV-GmERA1). Bars indicate SD. Asterisks at 0.3 and 0.5 h indicate a significant difference between ALSV-VC and ALSV-GmERA1 by <i>t</i>-test (*<i>P</i> < 0.05, **<i>P</i> < 0.01). (B) The surface temperature of detached fourth or fifth trifoliate leaflets of soybean plants inoculated with ALSV vectors was measured using thermography during the water-loss experiment shown in (A). Thermal images and photographs were taken 11 min after detachment. (C and D) Evaluation of leaf temperature (C) and stomatal conductance (D) in detached fourth or fifth trifoliate leaflets of soybean plants inoculated with ALSV vectors using a LI-6400XT portable photosynthesis system. Values are presented as mean ± SD (<i>n</i> = 3 for ALSV-VC and <i>n</i> = 4 for ALSV-GmERA1), and asterisks at 4, 8, and 12 min indicate a significant difference between ALSV-VC and ALSV-GmERA1 by <i>t</i>-test (**<i>P</i> < 0.01).</p

ALSV infection induces VIGS of <i>GmERA1s</i> in soybean.

<p>Silencing efficiency was evaluated by examining the expression of <i>GmERA1s</i> using quantitative RT-PCR and total RNAs derived from the fourth trifoliate leaves of soybean plants inoculated with the indicated inoculums. A conserved region in the two <i>GmERA1</i> genes was amplified by qRT-PCR. Relative values are presented as mean ± SD (<i>n</i> = 3) normalized to the expression of <i>GmACT11</i> (<i>Glyma15g05570</i>) as a control. Asterisks indicate a significant difference from ALSV-VC (empty vector) by <i>t</i>-test (*<i>P</i> < 0.05).</p

Down-regulation of <i>GmERA1s</i> enhances the stomatal closure response to ABA.

<p>Leaf disks from the fourth trifoliate leaves of ALSV-infected soybean plants were treated with or without ABA for 4 h. (A) Representative photographs of leaflets after ABA treatment (scale bars = 20 μm). (B) Stomatal aperture size data (shown as dots) from six independent measurements (≥60 stomata each). Bars indicate mean values. *<i>P</i> = 0.010, by <i>t</i>-test.</p

Evaluation of drought resistance in ALSV-GmERA1-infected soybean plants.

<p>Watering was withheld from plants at the V6 growth stage for 3 days. The drought status of the fourth to sixth trifoliate leaves was scored from 0 to 4 as follows: 0, no change; 1, wilting; 2, rolling; and 3, severe wilting. (A) Representative photographs of leaves with various scores. The leaves were scored at several time points after water withholding. (B) Leaf rolling score before rehydration (72–75 h after water withholding). The trial was repeated independently six times. (C) Plants were re-watered at 3 days after water withholding. Representative plants at one day after rehydration are shown.</p

Significance of light-induced hook exaggeration as reinforced by the concomitant anatomical change of germinating tomato seeds

Progression of the apical hook of tomato, Solanum lycopersicum, exaggerated by phytochrome mediation at the early germination stage, is followed in detail macroscopically and anatomically, and its proposed significance, i.e., survival by securing the seed coat release in the field, is reinforced by new findings. Furthermore, after self-release or artificial removal of the seed coat and the endosperm, no hook exaggeration occurs any more. Similar light-induced hook exaggeration (LIHE) is also found in carrot, parsley and Cryptotaenia japonica, which share some seed characteristics with tomato. These findings also support the above-stated significance