Cunyu Yan

DWARF27, an Iron-Containing Protein Required for the Biosynthesis of Strigolactones, Regulates Rice Tiller Bud Outgrowth

Tillering in rice (Oryza sativa) is one of the most important agronomic traits that determine grain yields. Previous studies on rice tillering mutants have shown that the outgrowth of tiller buds in rice is regulated by a carotenoid-derived MAX/RMS/D (more axillary branching) pathway, which may be conserved in higher plants. Strigolactones, a group of terpenoid lactones, have been recently identified as products of the MAX/RMS/D pathway that inhibits axillary bud outgrowth. We report here the molecular genetic characterization of d27, a classic rice mutant exhibiting increased tillers and reduced plant height. D27 encodes a novel iron-containing protein that localizes in chloroplasts and is expressed mainly in vascular cells of shoots and roots. The phenotype of d27 is correlated with enhanced polar auxin transport. The phenotypes of the d27 d10 double mutant are similar to those of d10, a mutant defective in the ortholog of MAX4/RMS1 in rice. In addition, 2′-epi-5-deoxystrigol, an identified strigolactone in root exudates of rice seedlings, was undetectable in d27, and the phenotypes of d27 could be rescued by supplementation with GR24, a synthetic strigolactone analog. Our results demonstrate that D27 is involved in the MAX/RMS/D pathway, in which D27 acts as a new member participating in the biosynthesis of strigolactones.

Insights into salt tolerance from the genome of Thellungiella salsuginea.

Thellungiella salsuginea, a close relative of Arabidopsis, represents an extremophile model for abiotic stress tolerance studies. We present the draft sequence of the T. salsuginea genome, assembled based on ∼134-fold coverage to seven chromosomes with a coding capacity of at least 28,457 genes. This genome provides resources and evidence about the nature of defense mechanisms constituting the genetic basis underlying plant abiotic stress tolerance. Comparative genomics and experimental analyses identified genes related to cation transport, abscisic acid signaling, and wax production prominent in T. salsuginea as possible contributors to its success in stressful environments.

A Small-Molecule Screen Identifies l-Kynurenine as a Competitive Inhibitor of TAA1/TAR Activity in Ethylene-Directed Auxin Biosynthesis and Root Growth in Arabidopsis

In this work, Kyn is identified as an auxin biosynthesis inhibitor that effectively and selectively targets TAA1-like Trp aminotransferases. Moreover, it describes a previously undiscovered positive feedback loop between auxin biosynthesis and ethylene signaling pathways in roots. The interactions between phytohormones are crucial for plants to adapt to complex environmental changes. One example is the ethylene-regulated local auxin biosynthesis in roots, which partly contributes to ethylene-directed root development and gravitropism. Using a chemical biology approach, we identified a small molecule, l-kynurenine (Kyn), which effectively inhibited ethylene responses in Arabidopsis thaliana root tissues. Kyn application repressed nuclear accumulation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Moreover, Kyn application decreased ethylene-induced auxin biosynthesis in roots, and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATEDs (TAA1/TARs), the key enzymes in the indole-3-pyruvic acid pathway of auxin biosynthesis, were identified as the molecular targets of Kyn. Further biochemical and phenotypic analyses revealed that Kyn, being an alternate substrate, competitively inhibits TAA1/TAR activity, and Kyn treatment mimicked the loss of TAA1/TAR functions. Molecular modeling and sequence alignments suggested that Kyn effectively and selectively binds to the substrate pocket of TAA1/TAR proteins but not those of other families of aminotransferases. To elucidate the destabilizing effect of Kyn on EIN3, we further found that auxin enhanced EIN3 nuclear accumulation in an EIN3 BINDING F-BOX PROTEIN1 (EBF1)/EBF2-dependent manner, suggesting the existence of a positive feedback loop between auxin biosynthesis and ethylene signaling. Thus, our study not only reveals a new level of interactions between ethylene and auxin pathways but also offers an efficient method to explore and exploit TAA1/TAR-dependent auxin biosynthesis.

Activation of the jasmonic acid pathway by depletion of the hydroperoxide lyase OsHPL3 reveals crosstalk between the HPL and AOS branches of the oxylipin pathway in rice.

The allene oxide synthase (AOS) and hydroperoxide lyase (HPL) branches of the oxylipin pathway, which underlie the production of jasmonates and aldehydes, respectively, function in plant responses to a range of stresses. Regulatory crosstalk has been proposed to exist between these two signaling branches; however, there is no direct evidence of this. Here, we identified and characterized a jasmonic acid (JA) overproduction mutant, cea62, by screening a rice T-DNA insertion mutant library for lineages that constitutively express the AOS gene. Map-based cloning was used to identify the underlying gene as hydroperoxide lyase OsHPL3. HPL3 expression and the enzyme activity of its product, (E)-2-hexenal, were depleted in the cea62 mutant, which resulted in the dramatic overproduction of JA, the activation of JA signaling, and the emergence of the lesion mimic phenotype. A time-course analysis of lesion formation and of the induction of defense responsive genes in the cea62 mutant revealed that the activation of JA biosynthesis and signaling in cea62 was regulated in a developmental manner, as was OsHPL3 activity in the wild-type plant. Microarray analysis showed that the JA-governed defense response was greatly activated in cea62 and this plant exhibited enhanced resistance to the T1 strain of the bacterial blight pathogen Xanthomonasoryzaepvoryzae (Xoo). The wounding response was attenuated in cea62 plants during the early stages of development, but partially recovered when JA levels were elevated during the later stages. In contrast, the wounding response was not altered during the different developmental stages of wild-type plants. These findings suggest that these two branches of the oxylipin pathway exhibit crosstalk with regards to biosynthesis and signaling and cooperate with each other to function in diverse stress responses.

Arabidopsis thaliana plants differentially modulate auxin biosynthesis and transport during defense responses to the necrotrophic pathogen Alternaria brassicicola

Although the role of auxin in biotrophic pathogenesis has been extensively studied, relatively little is known about its role in plant resistance to necrotrophs. Arabidopsis thaliana mutants defective in different aspects of the auxin pathway are generally more susceptible than wild-type plants to the necrotrophic pathogen Alternaria brassicicola. We show that A. brassicicola infection up-regulates auxin biosynthesis and down-regulates the auxin transport capacities of infected plants, these effects being partially dependent on JA signaling. We also show that these effects of A. brassicicola infection together lead to an enhanced auxin response in host plants. Application of IAA and MeJA together synergistically induces the expression of defense marker genes PDF1.2 (PLANT DEFENSIN 1.2) and HEL (HEVEIN-LIKE), suggesting that enhancement of JA-dependent defense signaling may be part of the auxin-mediated defense mechanism involved in resistance to necrotrophic pathogens. Our results provide molecular evidence supporting the hypothesis that JA and auxin interact positively in regulating plant resistance to necrotrophic pathogens and that activation of auxin signaling by JA may contribute to plant resistance to necrotrophic pathogens.

An in-advance stable isotope labeling strategy for relative analysis of multiple acidic plant hormones in sub-milligram Arabidopsis thaliana seedling and a single seed.

A sensitive and reliable in-advance stable isotope labeling strategy was developed for simultaneous relative quantification of 8 acidic plant hormones in sub-milligram amount of plant materials. Bromocholine bromide (BETA) and its deuterated counterpart D9-BETA were used to in-advance derivatize control and sample extracts individually, which were then combined and subjected to solid-phase extraction (SPE) purification followed by UPLC-MS/MS analysis. Relative quantification of target compounds was obtained by calculation of the peak area ratios of BETA/D9-BETA labeled plant hormones. The in-advance stable isotope labeling strategy realized internal standard-based relative quantification of multiple kinds of plant hormones independent of availability of internal standard of every analyte with enhanced sensitivity of 1-3 orders of magnitude. Meanwhile, the in-advance labeling contributes to higher sample throughput and more reliability. The method was successfully applied to determine 8 plant hormones in 0.8mg DW (dry weight) of seedlings and 4 plant hormones from single seed of Arabidopsis thaliana. The results show the potential of the method in relative quantification of multiple plant hormones in tiny plant tissues or organs, which will advance the knowledge of the crosstalk mechanism of plant hormones.

Hijacking of the jasmonate pathway by the mycotoxin fumonisin B1 (FB1) to initiate programmed cell death in Arabidopsis is modulated by RGLG3 and RGLG4

Highlight Two ubiquitin ligases control fumonisin B1-elicited programmed cell death by modulating jasmonate signalling transduction in Arabidopsis.

Differentiation of glucose-containing disaccharides isomers by fragmentation of the deprotonated non-covalent dimers using negative electrospray ionization tandem mass spectrometry

In this work, the glucose-containing disaccharide isomers were studied using negative electrospray ionization tandem mass spectrometry (ESI-MS/MS). Interestingly, the full-scan mass spectra of the disaccharides revealed that the deprotonated dimers were the predominant gas phase ions during ionization process. Importantly, several diagnostic fragment ions relative to linkage positions and anomeric configurations, arising from the covalent bond dissociation of dimers without breakdown of the non-covalent complexes, can be detected in the tandem mass spectra. Based on the scarce fragmentation characteristic, an original and simple approach for structural discrimination of disaccharide isomers was put forward. In addition, density functional theory (DFT) was employed to find out the reason why several fragmentations of intramolecular sugar bonds had preceded breakdown of the non-covalent complexes.

Analysis of Norditerpenoid Alkaloids Extracted from Aconitum sinomantanum Nakai by Electrospray Ionization Tandem Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was applied simultaneously in determining norditerpenoid alkaloids from the roots of Aconitum sinomantanum Nakai ( RAS) based on molecular mass information. The tandem mass spectra (ESI-MSn) provided the alkaloidal structural information, through which the existence of these alkaloids was further confirmed. Accordingly, six known norditerpenoid alkaloids were simultaneously determined on the basis of their ESI-MSn spectra. Furthermore, based on the diagnostic fragmentation pathways of alkaloidal MSn, a rapid method for direct detection and characterization of alkaloids from an ethanolic extract of RAS was described.

Gas phase isomeric differentiation of oleanolic and ursolic acids associated with heptakis‐(2,6‐di‐O‐methyl)‐β‐cyclodextrin by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Oleanolic acid (OA) and ursolic acid (UA) are isomeric triterpenoid compounds with similar pharmaceutical properties. Usually, modern chromatographic and electrophoretic methods are widely utilized to differentiate these two compounds. Compared with mass spectrometric (MS) methods, these modern separation methods are both time- and sample-consuming. Herein, we present a new method for structural differentiation of OA and UA by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) with the association of heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD). Exact MS and tandem MS (MS/MS) data showed that there is no perceptible difference between OA and UA, as well as their beta-cyclodextrin and gamma-cyclodextrin complexes. However, there is a remarkable difference in MS/MS spectra of DM-beta-CD complexes of OA and UA. The peak corresponding to the neutral loss of a formic acid and a water molecule could only be observed in the MS/MS spectrum of the complex of DM-beta-CD : OA. Molecular modeling calculations were also employed to further investigate the structural differences of DM-beta-CD : OA and DM-beta-CD : UA complexes. Therefore, by employing DM-beta-CD as a reference reagent, OA and UA could be differentiated with purely MS method.