Fu‐Yuan Zhu

In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues; however, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of short-read RNA sequencing, single molecule long-read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data from the two sequencing methods, approximately 83.4% of intron-containing genes were alternatively spliced. Two AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR); however, by contrast to AS events detected under normal conditions, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern, indicated by the increasing number of non-conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding of AS and alternative translation mechanisms under normal conditions, and in response to ABA treatment.

Indole acetic acid (IAA) is an important regulator of adventitious rooting via the activation of complex signaling cascades. In animals, carbon monoxide (CO), mainly generated by heme oxygenases (HOs), is a significant modulator of inflammatory reactions, affecting cell proliferation and the production of growth factors. In this report, we show that treatment with the auxin transport inhibitor naphthylphthalamic acid prevented auxin-mediated induction of adventitious rooting and also decreased the activity of HO and its by-product CO content. The application of IAA, HO-1 activator/CO donor hematin, or CO aqueous solution was able to alleviate the IAA depletion-induced inhibition of adventitious root formation. Meanwhile, IAA or hematin treatment rapidly activated HO activity or HO-1 protein expression, and CO content was also enhanced. The application of the HO-1-specific inhibitor zinc protoporphyrin IX (ZnPPIX) could inhibit the above IAA and hematin responses. CO aqueous solution treatment was able to ameliorate the ZnPPIX-induced inhibition of adventitious rooting. Molecular evidence further showed that ZnPPIX mimicked the effects of naphthylphthalamic acid on the inhibition of adventitious rooting, the down-regulation of one DnaJ-like gene (CSDNAJ-1), and two calcium-dependent protein kinase genes (CSCDPK1 and CSCDPK5). Application of CO aqueous solution not only dose-dependently blocked IAA depletion-induced inhibition of adventitious rooting but also enhanced endogenous CO content and up-regulated CSDNAJ-1 and CSCDPK1/5 transcripts. Together, we provided pharmacological, physiological, and molecular evidence that auxin rapidly activates HO activity and that the product of HO action, CO, then triggers the signal transduction events that lead to the auxin responses of adventitious root formation in cucumber (Cucumis sativus).

BACKGROUND: The next-generation sequencing (NGS) technology has greatly facilitated genomic and transcriptomic studies, contributing significantly in expanding the current knowledge on genome and transcriptome. However, the continually evolving variety of sequencing platforms, protocols and analytical pipelines has led the research community to focus on cross-platform evaluation and standardization. As a NGS pioneer in China, the Beijing Genomics Institute (BGI) has announced its own NGS platform designated as BGISEQ-500, since 2016. The capability of this platform in large-scale DNA sequencing and small RNA analysis has been already evaluated. However, the comparative performance of BGISEQ-500 platform in transcriptome analysis remains yet to be elucidated. The Illumina series, a leading sequencing platform in China's sequencing market, would be a preferable reference to evaluate new platforms. METHODS: WT (Col 0) transcriptome. The key parameters in RNA sequencing and transcriptomic data processing were assessed in biological replicate experiments, using aforesaid platforms. RESULTS: The results from the two platforms BGISEQ-500 and Illumina HiSeq4000 shared high concordance in both inter- (correlation, 0.88-0.93) and intra-platform (correlation, 0.95-0.98) comparison for gene quantification, identification of differentially expressed genes and alternative splicing events. However, the two platforms yielded highly variable interpretation results for single nucleotide polymorphism and insertion-deletion analysis. CONCLUSION: The present case study provides a comprehensive reference dataset to validate the capability of BGISEQ-500 enabling it to be established as a competitive and reliable platform in plant transcriptome analysis.

The rhizosheath, a layer of soil particles that adheres firmly to the root surface by a combination of root hairs and mucilage, may improve tolerance to drought stress. Setaria italica (L.) P. Beauv. (foxtail millet), a member of the Poaceae family, is an important food and fodder crop in arid regions and forms a larger rhizosheath under drought conditions. Rhizosheath formation under drought conditions has been studied, but the regulation of root hair growth and rhizosheath size in response to soil moisture remains unclear. To address this question, in this study we monitored root hair growth and rhizosheath development in response to a gradual decline in soil moisture. Here, we determined that a soil moisture level of 10%-14% (w/w) stimulated greater rhizosheath production compared to other soil moisture levels. Root hair density and length also increased at this soil moisture level, which was validated by measurement of the expression of root hair-related genes. These findings contribute to our understanding of rhizosheath formation in response to soil water stress.

Flavones are a major class of flavonoids with a wide range of physiological functions in plants. They are constitutively accumulated as C-glycosides and O-linked conjugates in vegetative tissues of grasses. It has long been presumed that the two structural modifications of flavones occur through independent metabolic routes. Previously, we reported that cytochrome P450 93G2 (CYP93G2) functions as a flavanone 2-hydroxylase (F2H) that provides 2-hydroxyflavanones for C-glycosylation in rice (Oryza sativa). Flavone C-glycosides are subsequently formed by dehydratase activity on 2-hydroxyflavanone C-glycosides. On the other hand, O-linked modifications were proposed to proceed after the flavone nucleus is generated. In this study, we demonstrate that CYP93G1, the closest homolog of CYP93G2 in rice, is a bona fide flavone synthase II (FNSII) that catalyzes the direct conversion of flavanones to flavones. In recombinant enzyme assays, CYP93G1 desaturated naringenin and eriodictyol to apigenin and luteolin, respectively. Consistently, transgenic expression of CYP93G1 in Arabidopsis (Arabidopsis thaliana) resulted in the accumulation of different flavone O-glycosides, which are not naturally present in cruciferous plants. Metabolite analysis of a rice CYP93G1 insertion mutant further demonstrated the preferential depletion of tricin O-linked flavanolignans and glycosides. By contrast, redirection of metabolic flow to the biosynthesis of flavone C-glycosides was observed. Our findings established that CYP93G1 is a key branch point enzyme channeling flavanones to the biosynthesis of tricin O-linked conjugates in rice. Functional diversification of F2H and FNSII in the cytochrome P450 CYP93G subfamily may represent a lineage-specific event leading to the prevalent cooccurrence of flavone C- and O-linked derivatives in grasses today.