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At least 19 records

Proteomic Insights into Trichome Responses to Elevated Elemental Stress in Cation Exchanger (CAX) Mutants

Abstract Research on elemental distribution in plants is crucial for understanding nutrient uptake, environmental adaptation and optimizing agricultural practices for sustainable food production. Plant trichomes, with their self-contained structures and easy accessibility, offer a robust model system for investigating elemental repartitioning. Transport proteins, such as the four functional cation exchangers (CAXs) in Arabidopsis, are low-affinity, high-capacity transporters primarily located on the vacuole. Mutants in these transporters have been partially characterized, one of the phenotypes of the CAX1 mutant being altered with tolerance to low-oxygen conditions. A simple visual screen demonstrated trichome density and morphology in cax1, and quadruple CAX (cax1-4: qKO) mutants remained unaltered. Here, we used synchrotron X-ray fluorescence (SXRF) to show that trichomes in CAX-deficient lines accumulated high levels of chlorine, potassium, calcium and manganese. Proteomic analysis on isolated Arabidopsis trichomes showed changes in protein abundance in response to changes in element accumulation. The CAX mutants showed an increased abundance of plasma membrane ATPase and vacuolar H-pumping proteins, and proteins associated with water movement and endocytosis, while also showing changes in proteins associated with the regulation of plasmodesmata. These findings advance our understanding of the integration of CAX transport with elemental homeostasis within trichomes and shed light on how plants modulate protein abundance under conditions of altered elemental levels.

59 BASIC BIOLOGICAL SCIENCES↗

Arabidopsis thaliana zinc accumulation in leaf trichomes is correlated with zinc concentration in leaves

Zinc (Zn) is a key micronutrient for plants and animals, and understanding Zn homeostasis in plants can improve both agriculture and human health. While root Zn transporters in plant model species have been characterized in detail, comparatively little is known about shoot processes controlling Zn concentrations and spatial distribution. Previous work showed that Zn hyperaccumulator species such as Arabidopsis halleri accumulate Zn and other metals in leaf trichomes. To date there is no systematic study regarding Zn accumulation in the trichomes of the non-accumulating, genetic model species A. thaliana. Here, we used Synchrotron X-Ray Fluorescence mapping to show that Zn accumulates at the base of trichomes of A. thaliana. Using transgenic and natural accessions of A thaliana that vary in bulk leaf Zn concentration, we demonstrate that higher leaf Zn increases total Zn found at the base of trichome cells. Our data indicates that Zn accumulation in trichomes is a function of the Zn status of the plant, and provides the basis for future studies on a genetically tractable plant species to understand the molecular steps involved in Zn spatial distribution in leaves.

59 BASIC BIOLOGICAL SCIENCES↗

Root hairs vs. trichomes: Not everyone is straight!

Trichomes show 47 morphological phenotypes, while literature reports only two root hair phenotypes in all plants. However, could hair-like structures exist below-ground in a similar wide range of morphologies like trichomes? Genetic mutants and root hair stress phenotypes point to the possibility of uncharacterized morphological variation existing belowground. For example, such root hairs in Arabidopsis (Arabidopsis thaliana) can be wavy, curled, or branched. We found hints in the literature about hair-like structures that emerge before root hairs belowground. As such, these early emerging hair structures can be potential exceptions to the contrasting morphological variation between trichomes and root hairs. Here, in this work, we show a previously unreported ‘hooked’ hair structure growing below-ground in common bean. The unique ‘hooking’ shape distinguishes the ‘hooked hair’ morphologically from root hairs. Currently, we cannot fully characterize the phenotype of our observation due to the lack of automated methods for phenotyping root hairs. This phenotyping bottleneck also handicaps the discovery of more morphology types that might exist below-ground as manual screening across species is slower than computer-assisted high-throughput screening.

59 BASIC BIOLOGICAL SCIENCES↗

Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA

As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.

59 BASIC BIOLOGICAL SCIENCES↗

Characterization of a Unique Pair of Ferredoxin and Ferredoxin NADP+ Reductase Isoforms That Operates in Non-Photosynthetic Glandular Trichomes

Our recent investigations indicated that isoforms of ferredoxin (Fd) and ferredoxin NADP+ reductase (FNR) play essential roles for the reductive steps of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway of terpenoid biosynthesis in peppermint glandular trichomes (GTs). Based on an analysis of several transcriptome data sets, we demonstrated the presence of transcripts for a leaf-type FNR (L-FNR), a leaf-type Fd (Fd I), a root-type FNR (R-FNR), and two root-type Fds (Fd II and Fd III) in several members of the mint family (Lamiaceae). The present study reports on the biochemical characterization of all Fd and FNR isoforms of peppermint (Mentha × piperita L.). The redox potentials of Fd and FNR isoforms were determined using photoreduction methods. Based on a diaphorase assay, peppermint R-FNR had a substantially higher specificity constant (kcat/Km) for NADPH than L-FNR. Similar results were obtained with ferricyanide as an electron acceptor. When assayed for NADPH–cytochrome c reductase activity, the specificity constant with the Fd II and Fd III isoforms (when compared to Fd I) was slightly higher for L-FNR and substantially higher for R-FNR. Based on real-time quantitative PCR assays with samples representing various peppermint organs and cell types, the Fd II gene was expressed very highly in metabolically active GTs (but also present at lower levels in roots), whereas Fd III was expressed at low levels in both roots and GTs. Our data provide evidence that high transcript levels of Fd II, and not differences in the biochemical properties of the encoded enzyme when compared to those of Fd III, are likely to support the formation of copious amounts of monoterpene via the MEP pathway in peppermint GTs. This work has laid the foundation for follow-up studies to further investigate the roles of a unique R-FNR–Fd II pair in non-photosynthetic GTs of the Lamiaceae.

59 BASIC BIOLOGICAL SCIENCES↗

Protein coating composition targets nanoparticles to leaf stomata and trichomes

Plant nanobiotechnology has the potential to revolutionize agriculture. However, the lack of effective methods to deliver nanoparticles (NPs) to the precise locations in plants where they are needed impedes these technological innovations. Here, model gold nanoparticles (AuNP) were coated with citrate, bovine serum albumin (BSA) as a protein control, or LM6-M, an antibody with an affinity for functional groups unique to stomata on leaf surfaces to deliver the AuNPs to stomata. One-month-old Vicia faba leaves were exposed via drop deposition to aqueous suspensions of LM6-M-coated AuNPs and allowed to air dry. After rinsing, Au distribution on the leaf surface was investigated by enhanced dark-field microscopy and X-ray fluorescence mapping. While citrate-coated AuNPs randomly covered the plant leaves, LM6M-AuNPs strongly adhered to the stomata and remained on the leaf surface after rinsing, and BSA-AuNPs specifically targeted trichome hairs. To the authors’ knowledge, this is the first report of active targeting of live leaf structures using NPs coated with molecular recognition molecules. Finally, this proof-of-concept study provides a strategy for future targeted nanopesticide delivery research.

60 APPLIED LIFE SCIENCES↗

Acetylation of homogalacturonan and rhamnogalacturonan‐I is catalyzed by a suite of trichome birefringence‐like proteins

SUMMARY Plant cell wall polysaccharides, including xylan, mannan, xyloglucan, and pectins, are often acetylated and members of the domain of unknown function 231 (DUF231)/trichome birefringence‐like (TBL) family have been shown to beO‐acetyltransferases mediating the acetylation of xylan, mannan, and xyloglucan. However, little is known about theO‐acetyltransferases responsible for pectin acetylation. In this report, we biochemically characterized a suite of Arabidopsis DUF231/TBL proteins for their roles in pectin acetylation. We generated 24 TBL recombinant proteins in mammalian cells and demonstrated that 10 of them were able to transfer acetyl groups from acetyl‐CoA onto the pectins homogalacturonan (HG) or rhamnogalacturonan‐I (RG‐I), and thus were named pectinO‐acetyltransferase 1 to 10 (POAT1 to 10). It was found that POAT2,4,9,10 specifically acetylated HG and POAT5,6 acetylated RG‐I, whereas POAT1,3,7,8 could act on both HG and RG‐I. The acetylation of HG and RG‐I by POATs was further corroborated by hydrolysis with pectin acetylesterases and by nuclear magnetic resonance spectroscopy. In addition, mutations of the conserved GDS and DXXH motifs in POAT3 and POAT8 were shown to lead to a loss of their ability to acetylate HG and RG‐I. Furthermore, simultaneous RNA interference downregulation ofPOAT1,3,6,7,8resulted in reduced cell expansion, impaired plant growth, and decreased pectin acetylation. Together, our findings indicate that these POATs are pectinO‐acetyltransferases involved in acetylation of the pectin polysaccharides HG and RG‐I.

Plant Sciences↗

Citric acid-assisted accumulation of Ni and other metals by Odontarrhena muralis: Implications for phytoextraction and metal foliar distribution assessed by μ-SXRF

Odontarrhena muralis is one of the most promissing plant species for Ni phytomining, and soil amendments can further increase its Ni phytoextraction ability. Here we investigated whether Ni phytomining/phytoremediation using this Ni hyperaccumulator can benefit from applying citric acid to a serpentine soil that is naturally enriched in Ni (>1000 mg kg -1 ). Synchrotron micro X-ray fluorescence (μ-SXRF) was used to image Ni and other metal distributions in whole fresh leaves of O. muralis. Leaf Ni accumulation in plants grown on citric acid-amended soil increased up to 55% while Co, Cr, Fe, Mn, and Zn concentrations were 4-, 14-, 6-, 7- and 1.3-fold higher than the control treatment. O. muralis presented high bioconcentration factors (leaf to soil concentration ratio) to Ni and Zn whereas Cr was seemingly excluded from uptake. The μ-SXRF images showed a uniform distribution of Ni, preferential localization of Co in the leaf tip, and clear concentration of Mn in the base of trichomes. The citric acid treatments strongly increased the Co fluoerescence intensity in the leaf tip and altered the spatial distribution of Mn across the leaf, but there was no difference in Ni fluorescence counts between the trichome-base region and the bulk leaf. Our data from a serpentine soil suggests that citrate treatment enhances Ni uptake, but Co is excreted from leaves even in low leaf concentrations, which can make Co phytoming using O. muralis unfeasible in natural serpentine soils.

54 ENVIRONMENTAL SCIENCES↗

Penetration of foliar-applied Zn and its impact on apple plant nutrition status: in vivo evaluation by synchrotron-based X-ray fluorescence microscopy

The absorption of foliar fertilizer is a complex process and is poorly understood. The ability to visualize and quantify the pathway that elements take following their application to leaf surfaces is critical for understanding the science and for practical applications of foliar fertilizers. By the use of synchrotron-based X-ray fluorescence to analyze the in vivo localization of elements, our study aimed to investigate the penetration of foliar-applied Zn absorbed by apple leaves with different physiological surface properties, as well as the possible interactions between foliar Zn level and the mineral nutrient status of treated leaves. The results indicate that the absorption of foliar-applied Zn was largely dependent on plant leaf surface characteristics. High-resolution elemental maps revealed that the high binding capacity of the cell wall for Zn contributed to the observed limitation of Zn penetration across epidermal cells. Trichome density and stomatal aperture had opposite effects on Zn fertilizer penetration: a relatively high density of trichomes increased the hydrophobicity of leaves, whereas the presence of stomata facilitated foliar Zn penetration. Low levels of Zn promoted the accumulation of other mineral elements in treated leaves, and the complexation of Zn with phytic acid potentially occurred owing to exposure to high-Zn conditions. The present study provides direct visual evidence for the Zn penetration process across the leaf surface, which is important for the development of strategies for Zn biofortification in crop species.

59 BASIC BIOLOGICAL SCIENCES↗

Engineering custom morpho- and chemotypes of Populus for sustainable production of biofuels, bioproducts, and biomaterials

Humans have been modifying plant traits for thousands of years, first through selection (i.e., domestication) then modern breeding, and in the last 30 years, through biotechnology. These modifications have resulted in increased yield, more efficient agronomic practices, and enhanced quality traits. Precision knowledge of gene regulation and function through high-resolution single-cell omics technologies, coupled with the ability to engineer plant genomes at the DNA sequence, chromatin accessibility, and gene expression levels, can enable engineering of complex and complementary traits at the biosystem level. Populus spp., the primary genetic model system for woody perennials, are among the fastest growing trees in temperate zones and are important for both carbon sequestration and global carbon cycling. Ample genomic and transcriptomic resources for poplar are available including emerging single-cell omics datasets. To expand use of poplar outside of valorization of woody biomass, chassis with novel morphotypes in which stem branching and tree height are modified can be fabricated thereby leading to trees with altered leaf to wood ratios. These morphotypes can then be engineered into customized chemotypes that produce high value biofuels, bioproducts, and biomaterials not only in specific organs but also in a cell-type-specific manner. For example, the recent discovery of triterpene production in poplar leaf trichomes can be exploited using cell-type specific regulatory sequences to synthesize high value terpenes such as the jet fuel precursor bisabolene specifically in the trichomes. By spatially and temporally controlling expression, not only can pools of abundant precursors be exploited but engineered molecules can be sequestered in discrete cell structures in the leaf. The structural diversity of the hemicellulose xylan is a barrier to fully utilizing lignocellulose in biomaterial production and by leveraging cell-type-specific omics data, cell wall composition can be modified in a tailored and targeted specific manner to generate poplar wood with novel chemical features that are amenable for processing or advanced manufacturing. Precision engineering poplar as a multi-purpose sustainable feedstock highlights how genome engineering can be used to re-imagine a crop species.

09 BIOMASS FUELS↗

Evolution of a plant gene cluster in Solanaceae and emergence of metabolic diversity

Plants produce phylogenetically and spatially restricted, as well as structurally diverse specialized metabolites via multistep metabolic pathways. Hallmarks of specialized metabolic evolution include enzymatic promiscuity and recruitment of primary metabolic enzymes and examples of genomic clustering of pathway genes. Solanaceae glandular trichomes produce defensive acylsugars, with sidechains that vary in length across the family. We describe a tomato gene cluster on chromosome 7 involved in medium chain acylsugar accumulation due to trichome specific acyl-CoA synthetase and enoyl-CoA hydratase genes. This cluster co-localizes with a tomato steroidal alkaloid gene cluster and is syntenic to a chromosome 12 region containing another acylsugar pathway gene. We reconstructed the evolutionary events leading to this gene cluster and found that its phylogenetic distribution correlates with medium chain acylsugar accumulation across the Solanaceae. This work reveals insights into the dynamics behind gene cluster evolution and cell-type specific metabolite diversity.

59 BASIC BIOLOGICAL SCIENCES↗

Predicting plant attractiveness to coccinellids with plant trait profiling, citizen science, and common garden surveys

Lady beetles are key predators in agricultural systems and their impact can be enhanced by plant resources. Facilitation of lady beetles is a potentially effective pest management strategy but a lack of data on which plant species attract and nurture them limits implementation. Here we investigate which plant species might optimize lady beetle impact by developing a profile of plant characteristics most likely to attract lady beetles and testing if the subset of “potentially preferred” plant families exhibiting those characteristics are significantly more attractive to lady beetles than other families. This testing encompassed a combination of observations submitted to a citizen science program, the Lost Ladybug Project, and two repeated site surveys. Traits reported to be most attractive to lady beetles included the presence of trichomes that provide protection from predation, nutritional resources such as nectar or prey, and visual or chemical cues associated with those traits. Significantly more lady beetles were observed on the three plant families which most often exhibit these traits, Apiaceae, Asteraceae, and Rosaceae, than on other families. Furthermore, strategies for incorporating plants from these families into production systems to attract lady beetles and limit pest damage are discussed.

59 BASIC BIOLOGICAL SCIENCES↗

Small subunits can determine enzyme kinetics of tobacco Rubisco expressed in Escherichia coli

Rubisco catalyses the first step in carbon fixation and is a strategic target to improve photosynthetic efficiency. In plants, Rubisco is composed of eight large and eight small subunits and its biogenesis requires multiple chaperones. We optimised a system to produce tobacco Rubisco in Escherichia coli by co-expressing chaperones in auto-induction medium. We successfully assembled tobacco Rubisco in E. coli with each small subunit that is normally encoded by the nuclear genome. Even though each enzyme carries only a single type of small subunit in E. coli, the enzymes exhibit carboxylation kinetics very similar to that of the native Rubisco. Tobacco Rubisco assembled with a recently discovered trichome small subunit has a higher catalytic rate and a lower CO 2 affinity than those assembled with other small subunits. Finally, our E. coli expression system will allow probing of features of both subunits of Rubisco that affect its kinetic properties.

59 BASIC BIOLOGICAL SCIENCES↗

Molecular Mechanism of Polysaccharide Acetylation by the Arabidopsis Xylan O-acetyltransferase XOAT1

Xylans are a major component of plant cell walls. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis (Arabidopsis thaliana) xylan O-acetyltransferase 1 (XOAT1), a member of the plant-specific Trichome Birefringence Like (TBL) family. Detailed characterization of XOAT1-catalyzed reactions by real-time NMR confirms that it exclusively catalyzes the 2-O-acetylation of xylan, followed by nonenzymatic acetyl migration to the O-3 position, resulting in products that are monoacetylated at both O-2 and O-3 positions. In addition, we report the crystal structure of the catalytic domain of XOAT1, which adopts a unique conformation that bears some similarities to the a/ß/a topology of members of the GDSL-like lipase/acylhydrolase family. Finally, we use a combination of biochemical analyses, mutagenesis, and molecular simulations to show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate, Ac–Ser-216, by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally uses an Arg residue in the formation of an oxyanion hole.

acetylation↗

Analysis of Dust and Corrosion Witness Samples Recovered from SNF Dry Storage Systems, Maine Yankee, 2023

This report documents the results of a long-term (5.79 year) exposure of 4-point bend corrosion test samples in the inlet and outlet vents of four spent nuclear fuel dry storage systems at the Maine Yankee Independent Spent Fuel Storage Installation. The goal of the test was to evaluate the corrosiveness of salt aerosols in a realistic near-marine environment, providing a data set for improved understanding of stress corrosion cracking of spent nuclear fuel dry storage canisters. Examination of the samples after extraction showed minor corrosion was present, mostly on rough-ground surfaces. However, dye penetrant testing showed that no SCC cracks were present. Dust collected on coupons co-located with the corrosion specimens was analyzed by scanning electron microscopy and leached to determine the soluble salts present. The dust was mostly organic material (pollen and stellate trichomes), with lesser detrital mineral grains. Salts present were a mix of sea-salts and continental salts, with chloride dominating the anions, but significant amounts of nitrate were also present. Both corrosion samples and dust samples showed evidence of wetting, indicating entry of water into the vents. The results of this field test suggest that the environment at Maine Yankee is not highly aggressive, although extrapolation from the periodically wetted vent samples to the hot, dry, canister surface may be difficult. No stress corrosion cracks were observed, but minor corrosion was present despite high nitrate concentrations in the salts. These observations may help address the ongoing question of the importance of nitrate in suppressing corrosion and SCC.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

Plant Cell Wall Polysaccharide O-Acetyltransferases

Plant cell walls are largely composed of polysaccharide polymers, including cellulose, hemicelluloses (xyloglucan, xylan, mannan, and mixed-linkage β-1,3/1,4-glucan), and pectins. Among these cell wall polysaccharides, xyloglucan, xylan, mannan, and pectins are often O-acetylated, and polysaccharide O-acetylation plays important roles in cell wall assembly and disease resistance. Genetic and biochemical analyses have implicated the involvement of three groups of proteins in plant cell wall polysaccharide O-acetylation: trichome birefringence-like (TBL)/domain of unknown function 231 (DUF231), reduced wall acetylation (RWA), and altered xyloglucan 9 (AXY9). Although the exact roles of RWAs and AXY9 are yet to be identified, members of the TBL/DUF231 family have been found to be O-acetyltransferases responsible for the O-acetylation of xyloglucan, xylan, mannan, and pectins. Here, we provide a comprehensive overview of the occurrence of O-acetylated cell wall polysaccharides, the biochemical properties, structural features, and evolution of cell wall polysaccharide O-acetyltransferases, and the potential biotechnological applications of manipulations of cell wall polysaccharide acetylation. Further in-depth studies of the biochemical mechanisms of cell wall polysaccharide O-acetylation will not only enrich our understanding of cell wall biology, but also have important implications in engineering plants with increased disease resistance and reduced recalcitrance for biofuel production.

Plant Sciences↗