Strigolactones play crucial roles in regulating plant architecture and development, as endogenous hormones, and orchestrating symbiotic interactions with fungi and parasitic plants, as components of root exudates. rac-GR24 is currently the most widely used strigolactone analog and serves as a reference compound in investigating the action of strigolactones. In this study, we evaluated a suite of debranones and found that 2-nitrodebranone (2NOD) exhibited higher biological activity than rac-GR24 in various aspects of plant growth and development in Arabidopsis, including hypocotyl elongation inhibition, root hair promotion and senescence acceleration. The enhanced activity of 2NOD in promoting AtD14-SMXL7 and AtD14-MAX2 interactions indicates that the molecular structure of 2NOD is a better match for the ligand perception site pocket of D14. Moreover, 2NOD showed lower activity than rac-GR24 in promoting Orobanche cumana seed germination, suggesting its higher ability to control plant architecture than parasitic interactions. In combination with the improved stability of 2NOD, these results demonstrate that 2NOD is a strigolactone analog that can specifically mimic the activity of strigolactones and that 2NOD exhibits strong potential as a tool for studying the strigolactone signaling pathway in plants.

UV-B light is a potential stress factor in plants, but how plants coordinate growth and UV-B stress responses is not well understood. Here, we report that brassinosteroid (BR) signaling inhibits UV-B stress responses in Arabidopsis (Arabidopsis thaliana) and various crops by controlling flavonol biosynthesis. We further demonstrate that BRI1-EMS-SUPPRESSOR 1 (BES1) mediates the tradeoff between plant growth and UV-B defense responses. BES1, a master transcription factor involved in BR signaling, represses the expression of transcription factor genes MYB11, MYB12, and MYB111, which activate flavonol biosynthesis. BES1 directly binds to the promoters of these MYBs in a BR-enhanced manner to repress their expression, thereby reducing flavonol accumulation. However, exposure to broadband UV-B down-regulates BES1 expression, thus promoting flavonol accumulation. These findings demonstrate that BR-activated BES1 not only promotes growth but also inhibits flavonoid biosynthesis. UV-B stress suppresses the expression of BES1 to allocate energy to flavonoid biosynthesis and UV-B stress responses, allowing plants to switch from growth to UV-B stress responses in a timely manner.

Purpose The expression of microRNA-505 (miR-505) has been investigated in various cancers; however, its effect and mechanism in relation to gastric cancer (GC) are yet to be determined. Thus, the current evaluation aimed to examine the expression and potential role of miR-505 in GC. Materials and methods Quantitative real-time PCR was carried out to analyze miR-505 expression in GC cells and tissues. We observed that miR-505 is differentially expressed in GC cells following transfection of its mimics or inhibitors. Changes in cell invasion, cell proliferation, and epithelial–mesenchymal transition markers were measured. Results These findings indicated that miR-505 expression is downregulated in both GC cell lines and GC tissues. In addition, knockdown miR-505 induced the invasion and proliferation of GC cells. Transfection of miR-505 mimics led to an elevation in N-cadherin expression but a decrease in E-cadherin expression. Furthermore, we have shown that miR-505 binds to the 3′-UTR region of Polo-like kinase-1. Conclusion Our results indicated that miR-505 suppresses GC cell proliferation and invasion; it may be a valuable candidate gene for seeking therapy strategy for GC.

UV-B light (UV-B radiation) is known to inhibit plant growth, but the mechanism is not well understood. UVR8 (UV RESISTANCE LOCUS 8) is a UV-B light photoreceptor that mediates UV-B light responses in plants. We report here that UV-B inhibits plant growth by repressing plant steroid hormone brassinosteroid (BR)-promoted plant growth. UVR8 physically interacts with the functional dephosphorylated BES1 (BRI1-EMS-SUPPRESSOR1) and BIM1 (BES1-INTERACTING MYC-LIKE 1) transcription factors that mediate BR-regulated gene expression and plant growth to inhibit their activities. Genome-wide gene expression analysis defined a BES1-dependent UV-B-regulated transcriptome, which is enriched with genes involved in cell elongation and plant growth. We further showed that UV-B-activated and nucleus-localized UVR8 inhibited the DNA-binding activities of BES1/BIM1 to directly regulate transcription of growth-related genes. Our results therefore establish that UVR8-BES1/BIM1 interaction represents an early photoreceptor signaling mechanism in plants and serves as an important module integrating light and BR signaling.

Jasmonate (JA) and its derivatives play an important role in plant development and defense. The JA signal receptor is an F-box protein COI1. COI1 combines with SKP1 and Cullin1 to form a SCF COI1 complex that interacts with JAZ repressors after combining with JA leading to ubiquitination and degradation of JAZ1 by 26S proteasome. JA-related transcriptional factors are released to control the downstream gene expression. Identification of COI1 is essential to understand the JA signaling pathway. However, expression and purification of bioactive COI1 protein is extremely difficult, limiting the study of further biological function. In this study, COI1 protein was fused with specific sequence tag and transferred into tobacco. After optimizing transformation time, and extraction procedure, the fusion protein was successfully isolated by affinity purification. Pull-down assay indicated that the purified protein interacted with JAZ1 present in JA-Ile, suggesting that the fusion protein was bioactive. The methodology developed in this study provides an efficient strategy to express and purify eukaryotic protein with bioactivity.

Strigolactones (SLs) are a group of carotenoid-derived terpenoid lactones with similar structures. They can promote symbiotic interactions of plants with soil microbes (Akiyama et al., 2010Akiyama K. Ogasawara S. Ito S. Hayashi H. Structural requirements of strigolactones for hyphal branching in AM fungi.Plant Cell Physiol. 2010; 51: 1104-1117Crossref PubMed Scopus (233) Google Scholar), stimulate the germination of parasitic weeds Striga and Orobanche (Cook et al., 1966Cook C.E. Whichard L.P. Turner B. Wall M.E. Germination of witchweed (Striga lutea Lour.): isolation and properties of a potent stimulant.Science. 1966; 154: 1189Crossref PubMed Scopus (695) Google Scholar, Johnson et al., 1976Johnson A.W. Roseberry G. Parker C.A. Novel approach to Striga and Orobanche control using synthetic germination stimulants.Weed Res. 1976; 16: 223-227Crossref Scopus (190) Google Scholar), and regulate plant growth and development, including inhibition of shoot (tiller) outgrowth (Gomez-Roldan et al., 2008Gomez-Roldan V. Fermas S. Brewer P.B. Puech-Pages V. Dun E.A. Pillot J.P. Letisse F. Matusova R. Danoun S. Portais J.C. et al.Strigolactone inhibition of shoot branching.Nature. 2008; 455: 189-194Crossref PubMed Scopus (1508) Google Scholar, Umehara et al., 2008Umehara M. Hanada A. Yoshida S. Akiyama K. Arite T. Takeda-Kamiya N. Magome H. Kamiya Y. Shirasu K. Yoneyama K. et al.Inhibition of shoot branching by new terpenoid plant hormones.Nature. 2008; 455: 195-200Crossref PubMed Scopus (1393) Google Scholar), suppression of hypocotyl elongation in Arabidopsis (Scaffidi et al., 2014Scaffidi A. Waters M.T. Sun Y.K. Skelton B.W. Dixon K.W. Ghisalberti E.L. Flematti G.R. Smith S.M. Strigolactone hormones and their stereoisomers signal through two related receptor proteins to induce different physiological responses in Arabidopsis.Plant Physiol. 2014; 165: 1221-1232Crossref PubMed Scopus (191) Google Scholar), and regulation of root architecture (Koltai, 2011Koltai H. Strigolactones are regulators of root development.New Phytol. 2011; 190: 545-549Crossref PubMed Scopus (158) Google Scholar). Recent studies revealed that SLs are recognized and hydrolyzed by an α/β-hydrolase protein DWARF14 (D14) to trigger the interaction of D14 with MORE AXILLARY GROWTH2 (MAX2) or with downstream repressors, such as SUPPRESSOR OF MORE AXILLARY GROWTH2-LIKE 6/7/8 (SMXL6/7/8) in Arabidopsis, which further leads to the degradation of repressors through the ubiquitin-proteasome system in a D14- and MAX2-dependent manner (Morffy et al., 2016Morffy N. Faure L. Nelson D.C. Smoke and hormone mirrors: action and evolution of karrikin and strigolactone signaling.Trends Genet. 2016; 32: 176-188Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Up to now, more than 20 kinds of natural SLs have been identified in the root exudates of many plant species, including cotton, rice, tobacco, and sorghum (Cook et al., 1966Cook C.E. Whichard L.P. Turner B. Wall M.E. Germination of witchweed (Striga lutea Lour.): isolation and properties of a potent stimulant.Science. 1966; 154: 1189Crossref PubMed Scopus (695) Google Scholar, Xie et al., 2013Xie X.N. Yoneyama K. Kisugi T. Uchida K. Ito S. Akiyama K. Hayashi H. Yokota T. Nomura T. Yoneyama K. Confirming stereochemical structures of strigolactones produced by rice and tobacco.Mol. Plant. 2013; 6: 153-163Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Studies on the different canonical strigolactone structures revealed that a conserved butenolide ring (D-ring) connected to a tricyclic lactone (ABC-ring) via an enol ether bridge forms the core structure of SLs, and three chiral carbon atoms in the BCD-ring are able to result in four different configurations of SLs. GR24 is a synthetic mimic molecule of 5-deoxystrigol and is widely used in SL studies. Previous studies found that 5-deoxystrigol (5DS), 4-deoxyorobanchol (4DO), and their corresponding enantiomers, as well as four stereoisomers of GR24, displayed different activities on inducing hyphal branches of germinating spores of the AM fungus Gigaspora margarita, demonstrating the importance of configurations of SLs for bioactivity (Akiyama et al., 2010Akiyama K. Ogasawara S. Ito S. Hayashi H. Structural requirements of strigolactones for hyphal branching in AM fungi.Plant Cell Physiol. 2010; 51: 1104-1117Crossref PubMed Scopus (233) Google Scholar). Recent research further revealed that different GR24 stereoisomers exhibited significantly diverse effects on hypocotyl length inhibition or shoot branching inhibition, suggesting that the (R) configuration at C-2′ in the D-ring is critical for inhibiting shoot branching in rice and Arabidopsis, and highlighting that the configuration of the BCD-ring is closely related to the biological functions of SLs (Scaffidi et al., 2014Scaffidi A. Waters M.T. Sun Y.K. Skelton B.W. Dixon K.W. Ghisalberti E.L. Flematti G.R. Smith S.M. Strigolactone hormones and their stereoisomers signal through two related receptor proteins to induce different physiological responses in Arabidopsis.Plant Physiol. 2014; 165: 1221-1232Crossref PubMed Scopus (191) Google Scholar, Umehara et al., 2015Umehara M. Cao M. Akiyama K. Akatsu T. Seto Y. Hanada A. Li W. Takeda-Kamiya N. Morimoto Y. Yamaguchi S. Structural requirements of strigolactones for shoot branching inhibition in rice and Arabidopsis.Plant Cell Physiol. 2015; 56: 1059-1072Crossref PubMed Scopus (68) Google Scholar). Here, we analyzed the biological effects of four GR24 stereoisomers (Figure 1A ) in various biochemical and physiological assays to investigate the SL configuration activity relationship. We first attempted to investigate whether Arabidopsis D14 (AtD14) protein has distinct hydrolysis activity on different GR24 stereoisomers. GR24 stereoisomers were incubated with purified recombinant AtD14 protein and subjected to quantification by HPLC-MS analysis. The presence of AtD14 in the reactions lowered the residual content of GR245DS to 29.3%, GR24ent-4DO to 92.6%, GR24ent-5DS to 39.5%, and GR244DO to 32.4%, compared with the control reaction without AtD14 (Figure 1B). The results showed that GR245DS and GR244DO are the most efficient substrates being hydrolyzed by AtD14, suggesting that the (R) configuration at C-2′ in the D-ring of SLs plays an important role in being hydrolyzed by AtD14. Furthermore, we performed an in vitro pull-down assay to assess the ability of GR24 stereoisomers on promoting the interaction of AtD14 with Arabidopsis MAX2 or with Arabidopsis SMXL7. Recombinant GST-AtD14 was incubated with resin-bound His-MAX2 or Flag-SMXL7 in the presence of GR24 stereoisomers, and then the recovery of AtD14 was detected by immunoblotting. The results showed that GR24 stereoisomers stimulated the recovery of AtD14 by MAX2 to various extents, with the relative order of activity being GR245DS > GR244DO > GR24ent-5DS > GR24ent-4DO (Figure 1C). Pull-down assays performed with SMXL7 showed that the specificity of GR24 stereoisomers on promoting the interaction of AtD14 with SMXL7 was identical to that with MAX2 (Figure 1D). Consistent with SMXL7, Dwarf 53 (D53) protein, a homolog of SMXL7 in rice, displayed the same interaction order in response to these GR24 stereoisomers (Supplemental Figure 1). Together with the results of a hydrolytic assay, these results demonstrated that GR245DS and GR244DO exhibited the highest activity to trigger SL signaling, followed by GR24ent-5DS and GR24ent-4DO, which highlighted the essential role of C-2′R structure in the D ring of SLs for signaling. These results also showed that rac-GR24, the commonly used GR24, exhibited moderate activity between GR245DS and GR24ent-5DS, in agreement with its constituent as a racemic mixture of these two stereoisomers. We further investigated the bioactivities of four GR24 stereoisomers with physiological assays. In the assay of inhibition of primary rosette shoot branching of the SL-deficient mutant max3 (Supplemental Figure 2), the bioactivities of GR24 stereoisomers are consistent with their activities in AtD14 hydrolytic and pull-down experiments, providing new evidence that shoot branching of Arabidopsis is primarily regulated by SL signaling. Furthermore, we compared the function of GR24 stereoisomers on Col-0 and SL-insensitive mutant max2 in inhibiting hypocotyl elongation at different concentrations (Figure 1E and Supplemental Figure 3) and in root development, including promotion of primary root elongation (Figure 1F) and inhibition of the density of lateral roots (Figure 1G). We found that, except for GR24ent-4DO, which was inactive, the other three GR24 stereoisomers stimulated elongation of the primary root length of Col-0 at relative low concentration (1 μM) (Figure 1F). In the other assays mentioned above, GR245DS showed the strongest activity, followed by GR244DO and GR24ent-5DS; GR24ent-4DO was the weakest, which was roughly consistent with the results of the hydrolytic and pull-down assays (Figure 1B–1D). Moreover, the physiological activities of these four GR24 stereoisomers are dependent on MAX2 (Figure 1E–1G and Supplemental Figure 3). In the root-hair elongation assay, when sucrose was deficient in culture medium, GR24ent-4DO always exhibited very low activity in promoting the root-hair length of Col-0 (Supplemental Figures 4A and 4B), which is consistent with the results of the above-mentioned assays. But surprisingly, once enough sucrose was supplemented in the culture, GR24ent-4DO showed a boosted activity in early stages. As shown in Figure 1H, the activity of GR24ent-4DO peaked at 48 h and was even close to that of GR245DS (Supplemental Figure 5A). After that, the activity growth of GR24ent-4DO in elongating root hairs turned gradually attenuated compared to the other GR24 stereoisomers. When 72 h was reached, the effect of GR24ent-4DO on promoting root-hair growth had lagged behind other stereoisomers (Figure 1H). Furthermore, we observed that GR24 stereoisomers cannot stimulate the root-hair growth of max2 in the presence or absence of sucrose (Supplemental Figures 4C, 5B and 5C), which demonstrated that the regulation of root hairs by GR24 stereoisomers is dependent on MAX2. These results suggest that a new time- and MAX2-dependent regulation mechanism of GR24ent-4DO on root-hair elongation needs to be discovered. Previous studies showed that multiple phytohormones and environmental factors regulate root-hair morphogenesis. Although it has been revealed that SL signaling cooperates with ethylene, auxin, and phosphorus signals to regulate root-hair elongation in Arabidopsis (Koltai, 2011Koltai H. Strigolactones are regulators of root development.New Phytol. 2011; 190: 545-549Crossref PubMed Scopus (158) Google Scholar), the crosstalk between SLs and sugar signals on root-hair development has not yet been elucidated. Our findings show that GR24ent-4DO was able to effectively regulate root-hair length for a period of time in the presence of sucrose, suggesting that sugar signal has an impact on SL signaling. It is possible that the sugar signal might affect configuration, absorption or perception of GR24ent-4DO in root hairs by direct or indirect way since GR24ent-4DO has the lowest bioactivity in our pull-down assays. Karrikins (KARs) are a type of butenolide molecule that exhibit a similar structure and highly similar signaling mechanisms to SLs. KARs can stimulate seed germination and inhibit hypocotyl elongation in a KARRIKIN INSENSITIVE 2 (KAI2)- and MAX2-dependent way (Morffy et al., 2016Morffy N. Faure L. Nelson D.C. Smoke and hormone mirrors: action and evolution of karrikin and strigolactone signaling.Trends Genet. 2016; 32: 176-188Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Previous studies showed that KAR and SL responses preferred different stereochemical structures of GR24. It is intriguing that GR24ent-5DS can alleviate primary seed dormancy of Arabidopsis seeds, a typical function of KAR signaling, and inhibit hypocotyl length significantly in a KAI2-dependent way (Scaffidi et al., 2014Scaffidi A. Waters M.T. Sun Y.K. Skelton B.W. Dixon K.W. Ghisalberti E.L. Flematti G.R. Smith S.M. Strigolactone hormones and their stereoisomers signal through two related receptor proteins to induce different physiological responses in Arabidopsis.Plant Physiol. 2014; 165: 1221-1232Crossref PubMed Scopus (191) Google Scholar), which shows that KAI2 preferentially mediates responses to GR24ent-5DS. Our results found that GR24ent-5DS also participates in SL signaling (Figure 1B–1D and Supplemental Figure 2). These studies suggest that GR24ent-5DS would be a good candidate for clarifying stereochemistry in KAR recognition and investigating the similarities between SL and KAR signaling in their ligand recognition mechanism, which would contribute to the discovery of the endogenous molecules recognized by KAI2 in planta and provide deeper insights into the crosstalk between KAR and SL signaling. This work was supported by the National Natural Science Foundation of China (grant no. 31421001).

Structural, biochemical, mass spectrometry and genetic analyses define Arabidopsis thaliana AtD14 as a non-canonical hormone receptor for strigolactone, which hydrolyses strigolactone into a covalently linked intermediate molecule and undergoes an open-to-closed state transition for interaction with D3 to trigger strigolactone signalling.

Traditional virus-induced gene silencing (VIGS) is a powerful virus-based short interfering RNA-mediated RNA silencing technique for plant functional genomics. Besides short interfering RNAs, microRNAs (miRNAs) have also been shown to regulate gene expression by RNA silencing in various organisms. However, plant virus-based miRNA silencing has not been reported. In addition, a number of plant miRNAs have been identified or predicted, while their functions are largely unknown. Thus, there is an urgent need for the development of new technologies to study miRNA function. Here, we report that a modified cabbage leaf-curl geminivirus vector can be used to express artificial and endogenous miRNAs in plants. Using this viral miRNA expression system, we demonstrate that VIGS using artificial miRNAs, dubbed as "MIR VIGS," was effective to silence the expression of endogenous genes, including PDS, Su, CLA1, and SGT1, in Nicotiana benthamiana. Silencing of SGT1 led to the loss of N-mediated resistance to Tobacco mosaic virus. Furthermore, using this viral miRNA expression system, we found that viral ectopic expression of endogenous miR156 and miR165 but not their mutants in N. benthamiana resulted in earlier abnormal developmental phenotypes, and expression of miR165 induced abnormal chlorotic spots on leaves. These results demonstrate that the cabbage leaf-curl geminivirus-based miRNA expression system can be utilized not only to specifically silence genes involved in general metabolism and defense but also to investigate the function of endogenous miRNAs in plants.

Traditional virus-induced gene silencing (VIGS) is a powerful virus-based short interfering RNA-mediated RNA silencing technique for plant functional genomics. Besides short interfering RNAs, microRNAs (miRNAs) have also been shown to regulate gene expression by RNA silencing in various organisms. However, plant virus-based miRNA silencing has not been reported. In addition, a number of plant miRNAs have been identified or predicted, while their functions are largely unknown. Thus, there is an urgent need for the development of new technologies to study miRNA function. Here, we report that a modified cabbage leaf-curl geminivirus vector can be used to express artificial and endogenous miRNAs in plants. Using this viral miRNA expression system, we demonstrate that VIGS using artificial miRNAs, dubbed as “MIR VIGS,” was effective to silence the expression of endogenous genes, including PDS, Su, CLA1, and SGT1, in Nicotiana benthamiana. Silencing of SGT1 led to the loss of N-mediated resistance to Tobacco mosaic virus. Furthermore, using this viral miRNA expression system, we found that viral ectopic expression of endogenous miR156 and miR165 but not their mutants in N. benthamiana resulted in earlier abnormal developmental phenotypes, and expression of miR165 induced abnormal chlorotic spots on leaves. These results demonstrate that the cabbage leaf-curl geminivirus-based miRNA expression system can be utilized not only to specifically silence genes involved in general metabolism and defense but also to investigate the function of endogenous miRNAs in plants.