Multiplexed in situ hybridization reveals distinct lineage identities for major and minor vein initiation during maize leaf development.

Leaves of flowering plants are characterized by diverse venation patterns. Patterning begins with the selection of vein-forming procambial initial cells from within the ground meristem of a developing leaf, a process which is considered to be auxin-dependent, and continues until veins are anatomically differentiated with functional xylem and phloem. At present, the mechanisms responsible for leaf venation patterning are primarily characterized in the model eudicot Arabidopsis thaliana which displays a reticulate venation network. However, evidence suggests that vein development may proceed via a different mechanism in monocot leaves where venation patterning is parallel. Here, we employed Molecular Cartography, a multiplexed in situ hybridization technique, to analyze the spatiotemporal localization of a subset of auxin-related genes and candidate regulators of vein patterning in maize leaves. We show how different combinations of auxin influx and efflux transporters are recruited during leaf and vein specification and how major and minor vein ranks develop with distinct identities. The localization of the procambial marker PIN1a and the spatial arrangement of procambial initial cells that give rise to major and minor vein ranks further suggests that vein spacing is prepatterned across the medio-lateral leaf axis prior to accumulation of the PIN1a auxin transporter. In contrast, patterning in the adaxial-abaxial axis occurs progressively, with markers of xylem and phloem gradually becoming polarized as differentiation proceeds. Collectively, our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves.

RIP-Seq analysis of non-PPR chloroplast editing factors reveals broad RNA interactions and enrichment of less efficiently translated RNAs by OZ1 and ORRM1 complexes.

C-to-U RNA editing in angiosperm chloroplasts requires a large suite of proteins bound together in the editosome. The editosome is comprised of PPR proteins, RIP/MORFs, OZ proteins, and ORRM proteins that physically interact in high molecular weight complexes. The specific functions of non-PPR editing factors in the editosome are unclear, however, specific subsets of editing sites are affected by absence of non-PPR editing factors. Unlike the PPR components of editosomes that have predictable nucleotide specificities, domains present in non-PPR editing factors make RNA associations difficult to predict. In this study, chloroplast extracts were isolated from juvenile maize seedlings. RNAs were immunoprecipitated using polyclonal antibodies targeting non-PPR editing factors RIP9, OZ1, and ORRM1. RNA libraries from duplicate experiments were compared. RIP9 was associated with most of the non-ribosomal RNA content of chloroplasts, consistent with a general binding function to PPR L-motifs and tethering of large ribonucleoprotein complexes. The breadth of RNA associations was greater than predicted and include mRNAs without predicted editing sites, tRNA sequences, and introns. OZ1 and ORRM1 were associated with a highly similar pool of RNAs that have a bias toward lower translational efficiency values in mature chloroplasts. Lower translational efficiency was also associated with the pool of edited RNAs compared to RNAs without editing sites. The unexpected breadth of interactions by non-PPR editing factors suggests the editosome is large, diverse, and associated with RNAs with lower relative translational efficiency in mature chloroplasts.

MINI-AC: inference of plant gene regulatory networks using bulk or single-cell accessible chromatin profiles.

Gene regulatory networks (GRNs) represent the interactions between transcription factors (TF) and their target genes. Plant GRNs control transcriptional programs involved in growth, development, and stress responses, ultimately affecting diverse agricultural traits. While recent developments in accessible chromatin (AC) profiling technologies make it possible to identify context-specific regulatory DNA, learning the underlying GRNs remains a major challenge. We developed MINI-AC (Motif-Informed Network Inference based on Accessible Chromatin), a method that combines AC data from bulk or single-cell experiments with TF binding site (TFBS) information to learn GRNs in plants. We benchmarked MINI-AC using bulk AC datasets from different Arabidopsis thaliana tissues and showed that it outperforms other methods to identify correct TFBS. In maize, a crop with a complex genome and abundant distal AC regions, MINI-AC successfully inferred leaf GRNs with experimentally confirmed, both proximal and distal, TF-target gene interactions. Furthermore, we showed that both AC regions and footprints are valid alternatives to infer AC-based GRNs with MINI-AC. Finally, we combined MINI-AC predictions from bulk and single-cell AC datasets to identify general and cell-type specific maize leaf regulators. Focusing on C4 metabolism, we identified diverse regulatory interactions in specialized cell types for this photosynthetic pathway. MINI-AC represents a powerful tool for inferring accurate AC-derived GRNs in plants and identifying known and novel candidate regulators, improving our understanding of gene regulation in plants.

Toward unveiling transcriptome dynamics and regulatory modules at the maternal/filial interface of developing maize kernel.

The basal region of maize (Zea mays) kernels, which includes the pedicel, placenta-chalazal, and basal endosperm transfer layers, serves as the maternal/filial interface for nutrient transfer from the mother plant to the developing seed. However, transcriptome dynamics of this maternal/filial interface remain largely unexplored. To address this gap, we conducted high-temporal-resolution RNA sequencing of the basal and upper kernel regions between 4 and 32 days after pollination and deeply analyzed transcriptome dynamics of the maternal/filial interface. Utilizing 790 specifically and highly expressed genes in the basal region, we performed the gene ontology (GO) term and weighted gene co-expression network analyses. In the early-stage basal region, we identified five MADS-box transcription factors (TFs) as hubs. Their homologs have been demonstrated as pivotal regulators at the maternal/filial interface of rice or Arabidopsis, suggesting their potential roles in maize kernel development. In the filling-stage basal region, numerous GO terms associated with transcriptional regulation and transporters are significantly enriched. Furthermore, we investigated the molecular function of three hub TFs. Through genome-wide DNA affinity purification sequencing combined with promoter transactivation assays, we suggested that these three TFs act as regulators of 10 basal-specific transporter genes involved in the transfer of sugars, amino acids, and ions. This study provides insights into transcriptomic dynamic and regulatory modules of the maternal/filial interface. In the future, genetic investigation of these hub regulators must advance our understanding of maternal/filial interface development and function.

A microRNA528-ZmLac3 module regulates low phosphate tolerance in maize.

MicroRNAs are known to play a crucial role in plant development and physiology and become a target for investigating the regulatory mechanism underlying plant low phosphate tolerance. ZmmiR528 has been shown to display significantly different expression levels between wild-type and low Pi-tolerant maize mutants. However, its functional role in maize low Pi tolerance remains unknown. In the present study, we studied the role and underlying molecular mechanism of miR528 in maize with low Pi tolerance. Overexpression of ZmmiR528 in maize resulted in impaired root growth, reduced Pi uptake capacity and compromised resistance to Pi deficiency. By contrast, transgenic maize plants suppressing ZmmiR528 expression showed enhanced low Pi tolerance. Furthermore, ZmLac3 and ZmLac5 which encode laccase were identified and verified as targets of ZmmiR528. ZmLac3 transgenic plants were subsequently generated and were also found to play key roles in regulating maize root growth, Pi uptake and low Pi tolerance. Furthermore, auxin transport was found to be potentially involved in ZmLac3-mediated root growth. Moreover, we conducted genetic complementary analysis through the hybridization of ZmmiR528 and ZmLac3 transgenic plants and found a favorable combination with breeding potential, namely anti-miR528:ZmLac3OE hybrid maize, which exhibited significantly increased low Pi tolerance and markedly alleviated yield loss caused by low Pi stress. Our study has thus identified a ZmmiR528-ZmLac3 module regulating auxin transport and hence root growth, thereby determining Pi uptake and ultimately low Pi tolerance, providing an effective approach for low Pi tolerance improvement through manipulating the expression of miRNA and its target in maize.

Plant nucleoside N-ribohydrolases: riboside binding and role in nitrogen storage mobilization.

Cells save their energy during nitrogen starvation by selective autophagy of ribosomes and degradation of RNA to ribonucleotides and nucleosides. Nucleosides are hydrolyzed by nucleoside N-ribohydrolases (nucleosidases, NRHs). Subclass I of NRHs preferentially hydrolyzes the purine ribosides while subclass II is more active towards uridine and xanthosine. Here, we performed a crystallographic and kinetic study to shed light on nucleoside preferences among plant NRHs followed by in vivo metabolomic and phenotyping analyses to reveal the consequences of enhanced nucleoside breakdown. We report the crystal structure of Zea mays NRH2b (subclass II) and NRH3 (subclass I) in complexes with the substrate analog forodesine. Purine and pyrimidine catabolism are inseparable because nucleobase binding in the active site of ZmNRH is mediated via a water network and is thus unspecific. Dexamethasone-inducible ZmNRH overexpressor lines of Arabidopsis thaliana, as well as double nrh knockout lines of moss Physcomitrium patents, reveal a fine control of adenosine in contrast to other ribosides. ZmNRH overexpressor lines display an accelerated early vegetative phase including faster root and rosette growth upon nitrogen starvation or osmotic stress. Moreover, the lines enter the bolting and flowering phase much earlier. We observe changes in the pathways related to nitrogen-containing compounds such as ß-alanine and several polyamines, which allow plants to reprogram their metabolism to escape stress. Taken together, crop plant breeding targeting enhanced NRH-mediated nitrogen recycling could therefore be a strategy to enhance plant growth tolerance and productivity under adverse growth conditions.

Dynamic transcriptome landscape of maize pericarp development.

As a maternal tissue, the pericarp supports and protects for other components of seed, such as embryo and endosperm. Despite the importance of maize pericarp in seed, the genome-wide transcriptome pattern throughout maize pericarp development has not been well characterized. Here, we developed RNA-seq transcriptome atlas of B73 maize pericarp development based on 21 samples from 5 days before fertilization (DBP5) to 32 days after fertilization (DAP32). A total of 25 346 genes were detected in programming pericarp development, including 1887 transcription factors (TFs). Together with pericarp morphological changes, the global clustering of gene expression revealed four developmental stages: undeveloped, thickening, expansion and strengthening. Coexpression analysis provided further insights on key regulators in functional transition of four developmental stages. Combined with non-seed, embryo, endosperm, and nucellus transcriptome data, we identified 598 pericarp-specific genes, including 75 TFs, which could elucidate key mechanisms and regulatory networks of pericarp development. Cell wall related genes were identified that reflected their crucial role in the maize pericarp structure building. In addition, key maternal proteases or TFs related with programmed cell death (PCD) were proposed, suggesting PCD in the maize pericarp was mediated by vacuolar processing enzymes (VPE), and jasmonic acid (JA) and ethylene-related pathways. The dynamic transcriptome atlas provides a valuable resource for unraveling the genetic control of maize pericarp development.

Mining genic resources regulating nitrogen-use efficiency based on integrative biological analyses and their breeding applications in maize and other crops.

Nitrogen (N) is an essential factor for limiting crop yields, and cultivation of crops with low nitrogen-use efficiency (NUE) exhibits increasing environmental and ecological risks. Hence, it is crucial to mine valuable NUE improvement genes, which is very important to develop and breed new crop varieties with high NUE in sustainable agriculture system. Quantitative trait locus (QTL) and genome-wide association study (GWAS) analysis are the most common methods for dissecting genetic variations underlying complex traits. In addition, with the advancement of biotechnology, multi-omics technologies can be used to accelerate the process of exploring genetic variations. In this study, we integrate the substantial data of QTLs, quantitative trait nucleotides (QTNs) from GWAS, and multi-omics data including transcriptome, proteome, and metabolome and further analyze their interactions to predict some NUE-related candidate genes. We also provide the genic resources for NUE improvement among maize, rice, wheat, and sorghum by homologous alignment and collinearity analysis. Furthermore, we propose to utilize the knowledge gained from classical cases to provide the frameworks for improving NUE and breeding N-efficient varieties through integrated genomics, systems biology, and modern breeding technologies.

Maize lipid droplet-associated protein 2 is recruited by a virus to enhance viral multiplication and infection through regulating cellular fatty acid metabolism.

Pathogen infection induces massive reprogramming of host primary metabolism. Lipid and fatty acid (FA) metabolism is generally disrupted by pathogens and co-opted for their proliferation. Lipid droplets (LDs) that play important roles in regulating cellular lipid metabolism are utilized by a variety of pathogens in mammalian cells. However, the function of LDs during pathogenic infection in plants remains unknown. We show here that infection by rice black streaked dwarf virus (RBSDV) affects the lipid metabolism of maize, which causes elevated accumulation of C18 polyunsaturated fatty acids (PUFAs) leading to viral proliferation and symptom development. The overexpression of one of the two novel LD-associated proteins (LDAPs) of maize (ZmLDAP1 and ZmLDAP2) induces LD clustering. The core capsid protein P8 of RBSDV interacts with ZmLDAP2 and prevents its degradation through the ubiquitin-proteasome system mediated by a UBX domain-containing protein, PUX10. In addition, silencing of ZmLDAP2 downregulates the expression of FA desaturase genes in maize, leading to a decrease in C18 PUFAs levels and suppression of RBSDV accumulation. Our findings reveal that plant virus may recruit LDAP to regulate cellular FA metabolism to promote viral multiplication and infection. These results expand the knowledge of LD functions and viral infection mechanisms in plants.

Use of GRF-GIF chimeras and a ternary vector system to improve maize (Zea mays L.) transformation frequency.

Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium-mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene-editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1-VIR2 virulence helper plasmid to improve T-DNA delivery, and/or expressing a fusion protein between a GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1-VIR2 helper vector with no effect on event quality regarding T-DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5- to 6.5-fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR-/Cas9-mediated gene editing. Our results demonstrate how a GRF-GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene-editing applications and molecular biology studies in maize.

Molecular basis of one-step methyl anthranilate biosynthesis in grapes, sweet orange, and maize.

Plants synthesize an array of volatile compounds, many of which serve ecological roles in attracting pollinators, deterring herbivores, and communicating with their surroundings. Methyl anthranilate (MeAA) is an anti-herbivory defensive volatile responsible for grape aroma that is emitted by several agriculturally relevant plants, including citrus, grapes, and maize. Unlike maize, which uses a one-step anthranilate methyltransferase (AAMT), grapes have been thought to use a two-step pathway for MeAA biosynthesis. By mining available transcriptomics data, we identified two AAMTs in Vitis vinifera (wine grape), as well as one ortholog in "Concord" grape. Many angiosperms methylate the plant hormone salicylic acid (SA) to produce methyl salicylate, which acts as a plant-to-plant communication molecule. Because the Citrus sinensis (sweet orange) SA methyltransferase can methylate both anthranilate (AA) and SA, we used this enzyme to examine the molecular basis of AA activity by introducing rational mutations, which identified several active site residues that increase activity with AA. Reversing this approach, we introduced mutations that imparted activity with SA in the maize AAMT, which uncovered different active site residues from those in the citrus enzyme. Sequence and phylogenetic analysis revealed that one of the Vitis AAMTs shares an ancestor with jasmonic acid methyltransferases, similar to the AAMT from strawberry (Frageria sp.). Collectively, these data demonstrate the molecular mechanisms underpinning AA activity across methyltransferases and identify one-step enzymes by which grapes synthesize MeAA.

Multi-omics-driven advances in the understanding of triacylglycerol biosynthesis in oil seeds.

Vegetable oils are rich sources of polyunsaturated fatty acids and energy as well as valuable sources of human food, animal feed, and bioenergy. Triacylglycerols, which are comprised of three fatty acids attached to a glycerol backbone, are the main component of vegetable oils. Here, we review the development and application of multiple-level omics in major oilseeds and emphasize the progress in the analysis of the biological roles of key genes underlying seed oil content and quality in major oilseeds. Finally, we discuss future research directions in functional genomics research based on current omics and oil metabolic engineering strategies that aim to enhance seed oil content and quality, and specific fatty acids components according to either human health needs or industrial requirements.

Jasmonate mimic modulates cell elongation by regulating antagonistic bHLH transcription factors via brassinosteroid signaling.

Lodging restricts growth, development, and yield formation in maize (Zea mays L.). Shorter internode length is beneficial for lodging tolerance. However, although brassinosteroids (BRs) and jasmonic acid (JA) are known to antagonistically regulate internode growth, the underlying molecular mechanism is still unclear. In this study, application of the JA mimic coronatine (COR) inhibited basal internode elongation at the jointing stage and repressed expression of the cell wall-related gene XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 1 (ZmXTH1), whose overexpression in maize plants promotes internode elongation. We demonstrated that the basic helix-loop-helix (bHLH) transcription factor ZmbHLH154 binds directly to the ZmXTH1 promoter and induces its expression, whereas the bHLH transcription factor ILI1 BINDING BHLH 1 (ZmIBH1) inhibits this transcriptional activation by forming a heterodimer with ZmbHLH154. Overexpressing ZmbHLH154 led to longer internodes, whereas zmbhlh154 mutants had shorter internodes than the wild type. The core JA-dependent transcription factors ZmMYC2-4 and ZmMYC2-6 interacted with BRASSINAZOLE RESISTANT 1 (ZmBZR1), a key factor in BR signaling, and these interactions eliminated the inhibitory effect of ZmBZR1 on its downstream gene ZmIBH1. Collectively, these results reveal a signaling module in which JA regulates a bHLH network by attenuating BR signaling to inhibit ZmXTH1 expression, thereby regulating cell elongation in maize.

ZmXYL modulates auxin-induced maize growth.

Plant architecture, lodging resistance, and yield are closely associated with height. In this paper, we report the identification and characterization of two allelic EMS-induced mutants of Zea mays, xyl-1, and xyl-2 that display dwarf phenotypes. The mutated gene, ZmXYL, encodes an α-xylosidase which functions in releasing xylosyl residue from a ß-1,4-linked glucan chain. Total α-xylosidase activity in the two alleles is significantly decreased compared to wild-type plants. Loss-of-function mutants of ZmXYL resulted in a decreased xylose content, an increased XXXG content in xyloglucan (XyG), and a reduced auxin content. We show that auxin has an antagonistic effect with XXXG in promoting cell divisions within mesocotyl tissue. xyl-1 and xyl-2 were less sensitive to IAA compared to B73. Based on our study, a model is proposed that places XXXG, an oligosaccharide derived from XyG and the substrate of ZmXYL, as having a negative impact on auxin homeostasis resulting in the dwarf phenotypes of the xyl mutants. Our results provide a insight into the roles of oligosaccharides released from plant cell walls as signals in mediating plant growth and development.

Combination of transcriptomic, proteomic, and degradomic profiling reveals common and distinct patterns of pathogen-induced cell death in maize.

Regulated cell death (RCD) is crucial for plant development, as well as in decision-making in plant-microbe interactions. Previous studies revealed components of the molecular network controlling RCD, including different proteases. However, the identity, the proteolytic network as well as molecular components involved in the initiation and execution of distinct plant RCD processes, still remain largely elusive. In this study, we analyzed the transcriptome, proteome, and N-terminome of Zea mays leaves treated with the Xanthomonas effector avrRxo1, the mycotoxin Fumonisin B1 (FB1), or the phytohormone salicylic acid (SA) to dissect plant cellular processes related to cell death and plant immunity. We found highly distinct and time-dependent biological processes being activated on transcriptional and proteome levels in response to avrRxo1, FB1, and SA. Correlation analysis of the transcriptome and proteome identified general, as well as trigger-specific markers for cell death in Zea mays. We found that proteases, particularly papain-like cysteine proteases, are specifically regulated during RCD. Collectively, this study characterizes distinct RCD responses in Z. mays and provides a framework for the mechanistic exploration of components involved in the initiation and execution of cell death.

Maize PIMT2 repairs damaged 3-METHYLCROTONYL COA CARBOXYLASE in mitochondria, affecting seed vigor.

PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) affects seed vigor by repairing damaged proteins. While PIMT is capable of isoaspartyl (isoAsp) repair in all proteins, those proteins most susceptible to isoAsp formation have not been well characterized, and the mechanisms by which PIMT affects seed vigor remain largely unknown. Using co-immunoprecipitation and LC-MS/MS, we found that maize (Zea mays) PIMT2 (ZmPIMT2) interacted predominantly with both subunits of maize 3-METHYLCROTONYL COA CARBOXYLASE (ZmMCC). ZmPIMT2 is specifically expressed in the maize embryo. Both mRNA and protein levels of ZmPIMT2 increased during seed maturation and declined during imbibition. Maize seed vigor was decreased in the zmpimt2 mutant line, while overexpression of ZmPIMT2 in maize and Arabidopsis thaliana increased seed vigor upon artificial aging. ZmPIMT2 was localized in the mitochondria, as determined by subcellular localization assays using maize protoplasts. ZmPIMT2 binding to ZmMCCα was confirmed by luciferase complementation tests in both tobacco (Nicotiana benthamiana) leaves and maize protoplasts. Knockdown of ZmMCCα decreased maize seed aging tolerance. Furthermore, overexpression of ZmPIMT2 decreased the accumulation of isoAsp of ZmMCCα protein in seed embryos that underwent accelerated aging treatment. Taken together, our results demonstrate that ZmPIMT2 binds ZmMCCα in mitochondria, repairs isoAsp damage, and positively affects maize seed vigor.

Raffinose positively regulates maize drought tolerance by reducing leaf transpiration.

Drought stress is one of the major constraints of global crop production. Raffinose, a non-reducing trisaccharide, has been considered to regulate positively the plant drought stress tolerance; however, evidence that augmenting raffinose production in leaves results in enhanced plant drought stress tolerance is lacking. The biochemical mechanism through which raffinose might act to mitigate plant drought stress remains unidentified. ZmRAFS encodes Zea mays RAFFINOSE SYNTHASE, a key enzyme that transfers galactose from the galactoside galactinol to sucrose for raffinose production. Overexpression of ZmRAFS in maize increased the RAFS protein and the raffinose content and decreased the water loss of leaves and enhanced plant drought stress tolerance. The biomass of the ZmRAFS overexpressing plants was similar to that of non-transgenic control plants when grown under optimal conditions, but was significantly greater than that of non-transgenic plants when grown under drought stress conditions. In contrast, the percentage of water loss of the detached leaves from two independent zmrafs mutant lines, incapable of synthesizing raffinose, was greater than that from null segregant controls and this phenomenon was partially rescued by supplementation of raffinose to detached zmrafs leaves. In addition, while there were differences in water loss among different maize lines, there was no difference in stomata density or aperture. Taken together, our work demonstrated that overexpression of the ZmRAFS gene in maize, in contrast to Arabidopsis, increased the raffinose content in leaves, assisted the leaf to retain water, and enhanced the plant drought stress tolerance without causing a detectable growth penalty.

Dynamic transcriptome landscape of developing maize ear.

Seed number and harvesting ability in maize (Zea mays L.) are primarily determined by the architecture of female inflorescence, namely the ear. Therefore, ear morphogenesis contributes to grain yield and as such is one of the key target traits during maize breeding. However, the molecular networks of this highly dynamic and complex grain-bearing inflorescence remain largely unclear. As a first step toward characterizing these networks, we performed a high-spatio-temporal-resolution investigation of transcriptomes using 130 ear samples collected from developing ears with length from 0.1 mm to 19.0 cm. Comparisons of these mRNA populations indicated that these spatio-temporal transcriptomes were clearly separated into four distinct stages stages I, II, III, and IV. A total of 23 793 genes including 1513 transcription factors (TFs) were identified in the investigated developing ears. During the stage I of ear morphogenesis, 425 genes were predicted to be involved in a co-expression network established by eight hub TFs. Moreover, 9714 ear-specific genes were identified in the seven kinds of meristems. Additionally, 527 genes including 59 TFs were identified as especially expressed in ear and displayed high temporal specificity. These results provide a high-resolution atlas of gene activity during ear development and help to unravel the regulatory modules associated with the differentiation of the ear in maize.

Biochemical and structural basis of polyamine, lysine and ornithine acetylation catalyzed by spermine/spermidine N-acetyl transferase in moss and maize.

Polyamines such as spermidine and spermine are essential regulators of cell growth, differentiation, maintenance of ion balance and abiotic stress tolerance. Their levels are controlled by the spermidine/spermine N1 -acetyltransferase (SSAT) via acetylation to promote either their degradation or export outside the cell as shown in mammals. Plant genomes contain at least one gene coding for SSAT (also named NATA for N-AcetylTransferase Activity). Combining kinetics, HPLC-MS and crystallography, we show that three plant SSATs, one from the lower plant moss Physcomitrium patens and two from the higher plant Zea mays, acetylate various aliphatic polyamines and two amino acids lysine (Lys) and ornithine (Orn). Thus, plant SSATs exhibit a broad substrate specificity, unlike more specific human SSATs (hSSATs) as hSSAT1 targets polyamines, whereas hSSAT2 acetylates Lys and thiaLys. The crystal structures of two PpSSAT ternary complexes, one with Lys and CoA, the other with acetyl-CoA and polyethylene glycol (mimicking spermine), reveal a different binding mode for polyamine versus amino acid substrates accompanied by structural rearrangements of both the coenzyme and the enzyme. Two arginine residues, unique among plant SSATs, hold the carboxyl group of amino acid substrates. The most abundant acetylated compound accumulated in moss was N6 -acetyl-Lys, whereas N5 -acetyl-Orn, known to be toxic for aphids, was found in maize. Both plant species contain very low levels of acetylated polyamines. The present study provides a detailed biochemical and structural basis of plant SSAT enzymes that can acetylate a wide range of substrates and likely play various roles in planta.

Root growth of monocotyledons and dicotyledons is limited by different tissues.

Plant growth and morphogenesis are determined by the mechanical properties of its cell walls. Using atomic force microscopy, we have characterized the dynamics of cell wall elasticity in different tissues in developing roots of several plant species. The elongation growth zone of roots of all species studied was distinguished by a reduced modulus of elasticity of most cell walls compared to the meristem or late elongation zone. Within the individual developmental zones of roots, there were also significant differences in the elasticity of the cell walls of the different tissues, thus identifying the tissues that limit root growth in the different species. In cereals, this is mainly the inner cortex, whereas in dicotyledons this function is performed by the outer tissues-rhizodermis and cortex. These differences result in a different behaviour of the roots of these species during longitudinal dissection. Modelling of longitudinal root dissection using measured properties confirmed the difference shown. Thus, the morphogenesis of monocotyledonous and dicotyledonous roots relies on different tissues as growth limiting, which should be taken into account when analyzing the localization of associated molecular events. At the same time, no matrix polysaccharide was found whose immunolabelling in type I or type II cell walls would predict their mechanical properties. However, assessment of the degree of anisotropy of cortical microtubules showed a striking correlation with the elasticity of the corresponding cell walls in all species studied.