Auxin and tryptophan homeostasis are facilitated by the ISS1/VAS1 aromatic aminotransferase in arabidopsis

Indole-3-acetic acid (IAA) plays a critical role in regulating numerous aspects of plant growth and development. While there is much genetic support for tryptophan-dependent (Trp-D) IAA synthesis pathways, there is little genetic evidence for tryptophan-independent (Trp-I) IAA synthesis pathways. Using Arabidopsis, we identified two mutant alleles of ISS1 ( I: ndole S: evere S: ensitive) that display indole-dependent IAA overproduction phenotypes including leaf epinasty and adventitious rooting. Stable isotope labeling showed that iss1, but not WT, uses primarily Trp-I IAA synthesis when grown on indole-supplemented medium. In contrast, both iss1 and WT use primarily Trp-D IAA synthesis when grown on unsupplemented medium. iss1 seedlings produce 8-fold higher levels of IAA when grown on indole and surprisingly have a 174-fold increase in Trp. These findings indicate that the iss1 mutant's increase in Trp-I IAA synthesis is due to a loss of Trp catabolism. ISS1 was identified as At1g80360, a predicted aromatic aminotransferase, and in vitro and in vivo analysis confirmed this activity. At1g80360 was previously shown to primarily carry out the conversion of indole-3-pyruvic acid to Trp as an IAA homeostatic mechanism in young seedlings. Our results suggest that in addition to this activity, in more mature plants ISS1 has a role in Trp catabolism and possibly in the metabolism of other aromatic amino acids. We postulate that this loss of Trp catabolism impacts the use of Trp-D and/or Trp-I IAA synthesis pathways.T32 AR059033 - NIAMS NIH HH

Arrhythmia Classification Based on Multi-Domain Feature Extraction for an ECG Recognition System

Automatic recognition of arrhythmias is particularly important in the diagnosis of heart diseases. This study presents an electrocardiogram (ECG) recognition system based on multi-domain feature extraction to classify ECG beats. An improved wavelet threshold method for ECG signal pre-processing is applied to remove noise interference. A novel multi-domain feature extraction method is proposed; this method employs kernel-independent component analysis in nonlinear feature extraction and uses discrete wavelet transform to extract frequency domain features. The proposed system utilises a support vector machine classifier optimized with a genetic algorithm to recognize different types of heartbeats. An ECG acquisition experimental platform, in which ECG beats are collected as ECG data for classification, is constructed to demonstrate the effectiveness of the system in ECG beat classification. The presented system, when applied to the MIT-BIH arrhythmia database, achieves a high classification accuracy of 98.8%. Experimental results based on the ECG acquisition experimental platform show that the system obtains a satisfactory classification accuracy of 97.3% and is able to classify ECG beats efficiently for the automatic identification of cardiac arrhythmias

Ni-Based Janus Pentagonal Monolayers as Promising Water-Splitting Photocatalysts

Photocatalysts which can efficiently promote water splitting to generate hydrogen without using sacrificial reagents and cocatalysts are highly desirable. In this study, on the basis of first-principles calculations, we predict that a series of Ni-based Janus monolayers with a pentagonal structure are promising photocatalysts, where the hydrogen evolution reaction can solely be driven by photon-excited electrons. The stability of the investigated monolayers is affirmed through energetic analysis, phonon band structure calculations, and ab initio molecular dynamics simulations. From the perspective of the photocatalytic process, their high absorption coefficients (∼105 cm–1) guarantee strong light absorption, their intrinsic electric fields generated by the Janus structure are beneficial to charge transfer, and their high catalytic activity speeds up the hydrogen evolution reaction. Moreover, strain engineering turns out to be effective for tuning band alignment and improving the catalytic performance. This study provides a new type of photocatalyst with high solar-to-hydrogen efficiency

In situ hybridization analysis of <i>PtrGXM1</i>, <i>PtrGXM2</i>, <i>PtrGXM3</i> and <i>PtrGXM4</i> mRNAs in <i>Populus</i> stems.

<p>Cross sections of stems were hybridized with digoxigenin-labeled antisense probes of <i>PtrGXM1</i> (A), <i>PtrGXM2</i> (B), <i>PtrGXM3</i> (C), and <i>PtrGXM4</i> (D) or the sense probe of <i>PtrGXM1</i> as the control (E). The hybridized sections were incubated with alkaline phosphatase-conjugated antibodies and the hybridization signals are shown as purple color. co, cortex; pf, phloem fiber; sx, secondary xylem. Bar in (A) = 160 µm for (A) to (E).</p

Detection of glucuronoxylan methyltransferase activity in <i>Populus</i> stem microsomes.

<p>Microsomes isolated from developing stems of <i>Populus trichocarpa</i> were incubated with the methyl donor, ¹⁴C-radiolabeled <i>S</i>-adenosylmethionine, and the GlcA-substituted Xyl₄ acceptor. The methyltransferase activity (CPM) was measured by the transfer of the radiolabeled methyl group onto the acceptor. Error bars in (A), (B) and (C) denote the se of two biological replicates. (A) <i>Populus</i> stem microsomes exhibit a methyltransferase activity toward the GlcA-substituted Xyl₄ acceptor. Microsomes (200 µg protein) were used for each reaction unless otherwise indicated. (B) Time course of the methyltransferase activity exhibited by the microsomes. (C) The methyltransferase activity increases with increasing amount of microsomes. (D) MALDI analysis of the reaction products catalyzed by the methyltransferase activity in <i>Populus</i> microsomes. Microsomes were incubated with the GlcA-substituted Xyl₄ acceptor in the absence (top panel) or presence (lower panel) of <i>S</i>-adenosylmethionine (SAM). The ions [M+Na]⁺ at <i>m/z</i> 745 and 759 correspond to the GlcA-substituted Xyl₄ acceptor [(GlcA)Xyl₄] and the methylated acceptor with an increase of 14 D [(MeGlcA)Xyl₄], respectively. The ions at <i>m/z</i> 767 and 781 are attributed to the doubly sodiated species [M+2Na]⁺ of (GlcA)Xyl₄ and (MeGlcA)Xyl₄, respectively. The identity of the ion at <i>m/z</i> 787 is not known.</p

Mutations of Arabidopsis TBL32 and TBL33 Affect Xylan Acetylation and Secondary Wall Deposition.

Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls

Subcellular localization of PtrGXM1, PtrGXM2, PtrGXM3, and PtrGXM4.

<p>PtrGXM proteins tagged with yellow fluorescent protein (YFP) were expressed in Arabidopsis protoplasts, and the fluorescence signals were detected with a laser confocal microscope. (A) PtrGXMs are membrane proteins with one transmembrane helix near the N-terminus as predicted by the TMHMM2.0 program. Inside, the cytoplasmic side of the membrane; outside, the noncytoplasmic side of the membrane. (B) and (C) An Arabidopsis protoplast (B; differential interference contrast image) expressing YFP alone showing the fluorescence signals throughout the cytoplasm (C). (D) to (G) An Arabidopsis protoplast (D) co-expressing PtrGXM1-YFP (E) and the Golgi-localized FRA8-CFP (F). (H) to (K) An Arabidopsis protoplast (H) co-expressing PtrGXM2-YFP (I) and FRA8-CFP (J). (L) to (O) An Arabidopsis protoplast (L) co-expressing PtrGXM3-YFP (M) and FRA8-CFP (N). (P) to (S) An Arabidopsis protoplast (P) co-expressing PtrGXM4-YFP (Q) and FRA8-CFP (R). Note the superimposition of the fluorescence signals of PtrGXM1-YFP (G), PtrGXM2-YFP (K), PtrGXM3-YFP (O), and PtrGXM4 (S) with the Golgi marker FRA8-CFP.</p

Biochemical properties of the methyltransferase activities exhibited by the recombinant PtrGXM proteins.

<p>Recombinant PtrGXM proteins were incubated with ¹⁴C-labeled <i>S</i>-adenosylmethionine and the (GlcA)Xyl₄ acceptor, and the methyltransferase activity was determined by the transfer of the radiolabeled methyl group onto the acceptor. Error bars denote the se of three independent assays. (A) Time course of the transfer of the methyl group onto the (GlcA)Xyl₄ acceptor. (B) The methyltransferase activity is protein concentration-dependent. (C) Effect of temperature on the methyltransferase activity.</p

Kinetic properties of the PtrGXM methyltransferase activities.

<p>Recombinant PtrGXM proteins were assayed for the methyltransferase activity in the presence of various concentrations of the (GlcA)Xyl₄ acceptor. The results were analyzed by Lineweaver-Burk plots to determine the <i>K</i>_m and <i>V</i>_max values.</p

MALDI spectra of the reaction products catalyzed by PtrGXM1, PtrGXM2, PtrGXM3, and PtrGXM4 recombinant proteins.

<p>Purified recombinant PtrGXMs were incubated with the GlcA-substituted Xyl₄ [(GlcA)Xyl₄] acceptor and the <i>S</i>-adenosylmethionine methyl donor and the reaction products were purified and subjected to MALDI analysis. Note the appearance of a new ion peak at <i>m/z</i> 759 corresponding to (MeGlcA)Xyl₄ (red arrows) with an increase of 14 D relative to the (GlcA)Xyl₄ acceptor (<i>m/z</i> 745) in the products of reactions incubated with PtrGXMs but not with MBP. The ions at <i>m/z</i> 767 and 781 are attributed to the doubly sodiated species [M+2Na]⁺ of (GlcA)Xyl₄ and (MeGlcA)Xyl₄, respectively.</p