New Wine in an Old Bottle: Utilizing Chemical Genetics to Dissect Apical Hook Development

The apical hook is formed by dicot seedlings to protect the tender shoot apical meristem during soil emergence. Regulated by many phytohormones, the apical hook has been taken as a model to study the crosstalk between individual signaling pathways. Over recent decades, the roles of different phytohormones and environmental signals in apical hook development have been illustrated. However, key regulators downstream of canonical hormone signaling have rarely been identified via classical genetics screening, possibly due to genetic redundancy and/or lethal mutation. Chemical genetics that utilize small molecules to perturb and elucidate biological processes could provide a complementary strategy to overcome the limitations in classical genetics. In this review, we summarize current progress in hormonal regulation of the apical hook, and previously reported chemical tools that could assist the understanding of this complex developmental process. We also provide insight into novel strategies for chemical screening and target identification, which could possibly lead to discoveries of new regulatory components in apical hook development, or unidentified signaling crosstalk that is overlooked by classical genetics screening

Contribution of methylation regulation of MpDREB2A promoter to drought resistance of Mauls prunifolia

Background and aims: Malus prunifolia (Chinese name: Fu Ping Qiu Zi), a wild relative of cultivated apple (Malus x domestica Borkh), is extremely resistant to drought compared with domesticated cultivars, such as ‘Golden Delicious’. However, the molecular mechanisms underlying drought resistance of M. prunifolia have not been characterized. This study investigates a new regulatory mechanism to improve apple drought resistance. Methods: M. prunifolia and ‘Golden Delicious’ were each grafted on M. hupehensis for gene expression analysis. The methylation level of the DREB2A promoter was determined by bisulfite sequencing and ChIP-qPCR. Chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify target genes of MpDREB2A in apple. Results: The exposure to drought stress stimulated the expression level of DREB2A gene more than 100-fold in M. prunifolia, but only 16-fold in ‘Golden Delicious’. This difference in gene expression could not be explained in terms of difference in leaf relative water content. Correspondingly, the methylation level of M. prunifolia DREB2A (MpDREB2A) promoter region was significantly reduced. Additionally, MpDREB2A conferred enhanced drought resistance when ectopically expressed in Arabidopsis. Over 2800 potential downstream target genes of MpDREB2A were identified by ChIP-seq and these downstream genes have diverse potential functions related to stress resistance. Conclusions: Methylation regulation in promoter of MpDREB2A may contribute to the drought resistance of M. prunifolia.</p

Maximum Somatic Allele Frequency-Adjusted Blood-Based Tumor Mutational Burden Predicts the Efficacy of Immune Checkpoint Inhibitors in Advanced Non-Small Cell Lung Cancer

Introduction: Recent studies exhibited the unstable prediction ability of blood-based tumor mutational burden (bTMB) when predicting the response of immune checkpoint inhibitors (ICIs) therapy in patients with non-small cell lung cancer (NSCLC). Circulating tumor DNA (ctDNA) abundance, usually represented by maximum somatic allele frequency (MSAF), was one possible confounding factor influencing bTMB ability in ICIs response prediction. Methods: MSAF-adjusted bTMB (Ma-bTMB) was established and validated in patients with advanced NSCLC among Geneplus Cancer Genome Database (GCGD, n = 1679), Zhuo (n = 35), Wang (n = 45), POPLAR (NCT01903993, n = 211) and OAK (NCT02008227, n = 642) cohorts. Results: MSAF demonstrated a modest positive correlation with bTMB and a negative one with survival benefit. Improved survival outcomes of ICIs therapy have been observed among patients with high-Ma-bTMB compared to those with low-Ma-bTMB in Zhuo and Wang cohorts. In addition, compared to low-Ma-bTMB, high-Ma-bTMB was associated with more positive clinical benefits from ICIs therapy than chemotherapy both in POPLAR and OAK cohorts. Further exploration suggested that Ma-bTMB could precisely identify more potential ICIs beneficiaries compared to bTMB and LAF-bTMB, complementary to PD-L1 expression. Conclusions: We developed Ma-bTMB, a convenient, readily available, non-invasive predictive biomarker effectively differentiates beneficiaries of ICIs therapy in advanced NSCLC, warranting future clinical trials

An atypical R2R3 MYB transcription factor increases cold hardiness by CBF-dependent and CBF-independent pathways in apple

Apple (Malus × domestica) trees are vulnerable to freezing temperatures. However, there has been only limited success in developing cold-hardy cultivars. This lack of progress is due at least partly to lack of understanding of the molecular mechanisms of freezing tolerance in apple. In this study, we evaluated the potential roles for two R2R3 MYB transcription factors (TFs), MYB88 and the paralogous FLP (MYB124), in cold stress in apple and Arabidopsis. We found that MYB88 and MYB124 positively regulate freezing tolerance and cold-responsive gene expression in both apple and Arabidopsis. Chromatin-Immunoprecipitation-qPCR and electrophoretic mobility shift assays showed that MdMYB88/MdMYB124 act as direct regulators of the COLD SHOCK DOMAIN PROTEIN 3 (MdCSP3) and CIRCADIAN CLOCK ASSOCIATED 1 (MdCCA1) genes. Dual luciferase reporter assay indicated that MdCCA1 but not MdCSP3 activated the expression of MdCBF3 under cold stress. Moreover, MdMYB88 and MdMYB124 promoted anthocyanin accumulation and H2O2 detoxification in response to cold. Taken together, our results suggest that MdMYB88 and MdMYB124 positively regulate cold hardiness and cold-responsive gene expression under cold stress by C-REPEAT BINDING FACTOR (CBF)-dependent and CBF-independent pathways

Chemical genetic screening identifies nalacin as an inhibitor of GH3 amido synthetase for auxin conjugation

Auxin inactivation is critical for plant growth and development. To develop plant growth regulators functioning in auxin inactivation pathway, we performed a phenotype-based chemical screen in Arabidopsis and identified a chemical, nalacin, that partially mimicked the effects of auxin. Genetic, pharmacological, and biochemical approaches demonstrated that nalacin exerts its auxin-like activities by inhibiting indole-3-acetic acid (IAA) conjugation that is mediated by Gretchen Hagen 3 (GH3) acyl acid amido synthetases. The crystal structure of Arabidopsis GH3.6 in complex with D4 (a derivative of nalacin) together with docking simulation analysis revealed the molecular basis of the inhibition of group II GH3 by nalacin. Sequence alignment analysis indicated broad bioactivities of nalacin and D4 as inhibitors of GH3s in vascular plants, which were confirmed, at least, in tomato and rice. In summary, our work identifies nalacin as a potent inhibitor of IAA conjugation mediated by group II GH3 that plays versatile roles in hormone-regulated plant development and has potential applications in both basic research and agriculture