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The aln-3 mutant overaccumulating allantoin and respective wild type (WT) strain of Arabidopsis thaliana were exposed to cadmium (Cd) or mercury (Hg) with or without nitric oxide (NO) donor (sodium nitroprusside, SNP) to study cross-talk, metabolic and oxidative changes between these nitrogen sources (organic vs. inorganic). The aln-3 accumulated over 10-fold more allantoin than WT with the effect of Cd and Hg differing in leaf and root tissue: aln-3 contained more ascorbic acid and phytochelatins when treated with Cd or Hg and more Cd in both organs. SNP depleted leaf Cd and root Hg accumulation in aln3 but had a positive impact on the amount of metabolites typically in WT plants, indicating potentially negative relation between allantoin and NO. In agreement, aln-3 roots showed lower NO signals in control or metal treatments, but higher ROS signal, and SNP had more pronounced impact in WT roots. Flavonol glycosides were more abundant in aln-3 and were affected more by metals than by SNP. Malate was the most affected Krebs acid with strong reaction to SNP and Hg treatment. Data indicate that allantoin overaccumulation influences the accumulation of specific metabolites but nitric oxide has a greater impact on the metabolite profile in WT., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Plant pathogen effector proteins are key to pathogen virulence. In susceptible host Brassicas, the clubroot pathogen, Plasmodiophora brassicae , induces the production of nutrient-sink root galls, at the site of infection. Among a list of 32 P. brassiae effector candidates previously reported by our group, we identified SSPbP53 as a putative apoplastic cystatin-like protein highly expressed during the secondary infection. Here we found that SSPbP53 encoding gene is conserved among several P. brassicae pathotypes and that SSPbP53 is an apoplastic protein able to directly interact with and inhibit cruciferous papain-like cysteine proteases (PLCPs), specifically Arabidopsis XYLEM CYSTEINE PEPTIDASE 1 ( At XCP1). The severity of clubroot disease is greatly reduced in the Arabidopsis xcp1 null mutant ( At Δ xcp1 ) after infection with P. brassicae resting spores, indicating that the interaction of P. brassicae SSPbP53 with XCP1 is important to clubroot susceptibility. SSPbP53 is the first cystatin-like effector identified and characterized for a plant pathogenic protist.

Plasmodiophora brassicae (Wor.) is an obligate intracellular plant pathogen affecting Brassicas worldwide. Identification of effector proteins is key to understanding the interaction between P. brassicae and its susceptible host plants. To date, there is very little information available on putative effector proteins secreted by P. brassicae during a secondary infection of susceptible host plants, resulting in root gall production. A bioinformatics pipeline approach to RNA-Seq data from Arabidopsis thaliana (L.) Heynh. root tissues at 17, 20, and 24 d postinoculation (dpi) identified 32 small secreted P. brassicae proteins (SSPbPs) that were highly expressed over this secondary infection time frame. Functional signal peptides were confirmed for 31 of the SSPbPs, supporting the accuracy of the pipeline designed to identify secreted proteins. Expression profiles at 0, 2, 5, 7, 14, 21, and 28 dpi verified the involvement of some of the SSPbPs in secondary infection. For seven of the SSPbPs, a functional domain was identified using Blast2GO and 3D structure analysis and domain functionality was confirmed for SSPbP22, a kinase localized to the cytoplasm and nucleus., (© 2020 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.)

Allantoin, an important intermediate of ureide metabolism, has been the subject of investigation recently due to its dual function in nitrogen recycling and abiotic stress response in plants. Allantoin appears to be the dominant ureide accumulating in response to different abiotic stresses, and mutants containing elevated allantoin concentrations exhibit a stress-tolerant phenotype due to limited reactive oxygen species (ROS) generation. Here we describe the involvement of allantoin in stress response and attempt to explain the regulatory mechanism(s) underlying allantoin function in plants. Growth of wild type Col-0 seedlings in the presence of exogenous allantoin improved root elongation in response to Cd treatment. Allantoin treatment of Col-0 seeds increases superoxide dismutase activity causing an enhanced seed germination and seedling growth following Cd exposure. Additionally, allantoinase-overexpressed (ALNox) lines, with lower levels of allantoin, exhibited more susceptibility to Cd treatment than Col-0 Arabidopsis, implying that there is a positive correlation between allantoin concentration and Cd resistance in plants. Growing ABA-insensitive (abi) mutants on allantoin-containing media and comparison between abi mutants and their wild-type backgrounds demonstrated that the potential regulatory function of allantoin does not require ABA at germination but may be ABA-dependent at later stages of seedling growth, suggesting a potential crosstalk between allantoin-mediated stress response and ABA signalling pathway in plants., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Allantoin is a nitrogenous compound derived from purine catabolism that contributes to nitrogen recycling in plants. Accumulation of allantoin in plant tissues and a potential role in protection of plants from abiotic stress conditions has been identified. The present work shows that application of exogenous allantoin increased stress tolerance of Arabidopsis seedlings when germinated on, or subjected to the media containing NaCl. Allantoin-induced tolerance to NaCl stress was associated with decreased production of superoxide and hydrogen peroxide in seedlings. To understand the molecular mechanism, the effect of exogenous allantoin treatment on expression of several stress-related genes was investigated. Exogenous allantoin altered the expression of several antioxidant encoding genes and upregulated the expression of two genes involved in oxidative stress tolerance, SOS1 and RCD1, in the presence or absence of NaCl. Allantoin increased the NaCl tolerance of abscisic acid insensitive mutants, suggesting that it can function independently of abscisic acid signaling. These results provide additional evidence for the role of allantoin in enhancing plants tolerance to oxidative stress., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Background: Clubroot is an important disease caused by the obligate parasite Plasmodiophora brassicae that infects the Brassicaceae. As a soil-borne pathogen, P. brassicae induces the generation of abnormal tissue in the root, resulting in the formation of galls. Root infection negatively affects the uptake of water and nutrients in host plants, severely reducing their growth and productivity. Many studies have emphasized the molecular and physiological effects of the clubroot disease on root tissues. The aim of the present study is to better understand the effect of P. brassicae on the transcriptome of both shoot and root tissues of Arabidopsis thaliana., Results: Transcriptome profiling using RNA-seq was performed on both shoot and root tissues at 17, 20 and 24 days post inoculation (dpi) of A. thaliana, a model plant host for P. brassicae. The number of differentially expressed genes (DEGs) between infected and uninfected samples was larger in shoot than in root. In both shoot and root, more genes were differentially regulated at 24 dpi than the two earlier time points. Genes that were highly regulated in response to infection in both shoot and root primarily were involved in the metabolism of cell wall compounds, lipids, and shikimate pathway metabolites. Among hormone-related pathways, several jasmonic acid biosynthesis genes were upregulated in both shoot and root tissue. Genes encoding enzymes involved in cell wall modification, biosynthesis of sucrose and starch, and several classes of transcription factors were generally differently regulated in shoot and root., Conclusions: These results highlight the similarities and differences in the transcriptomic response of above- and below-ground tissues of the model host Arabidopsis following P. brassicae infection. The main transcriptomic changes in root metabolism during clubroot disease progression were identified. An overview of DEGs in the shoot underlined the physiological changes in above-ground tissues following pathogen establishment and disease progression. This study provides insights into host tissue-specific molecular responses to clubroot development and may have applications in the development of clubroot markers for more effective breeding strategies.

Ureides are nitrogen-rich compounds, derived from purine catabolism. A dual role for ureides, and for allantoin in particular, in both nitrogen recycling and the abiotic stress response has been recently identified. Previous work on the effect of allantoin on cadmium (Cd)-exposed Arabidopsis revealed that high concentration of allantoin in allantoinase-negative mutant (aln-3) leaves alleviates Cd toxicity via inducing antioxidant mechanisms in these plants. In the present study, we evaluate whether allantoin has a similar protective role in roots. Both wild type and aln-3 roots contain higher amounts of internal Cd compared to leaves. Likewise, aln-3 roots are more resistant to Cd, reflected in fresh and dry weight, and stimulated antioxidant enzyme activity, including superoxide dismutase (SOD) and catalase (CAT), resulting in lower reactive oxygen species concentration. In contrast with wild-type leaves, high levels of Cd in Col-0 roots reduces transcript abundance of uricase, leading to a significant decline in allantoin level of treated roots at 1000 and 1500 μM CdCl 2 . This metabolite change is also accompanied by decreasing the activity of antioxidant enzymes (SOD and CAT). Additionally, contrary to wild-type leaves, root genotype has a significant effect on CAT activity under Cd treatment, suggesting the possible different sources of damage and oxidative stress response in these two tissues., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Plants apply various molecular, physiological and morphological strategies in response to undesirable environmental conditions. One of the possible responses which may contribute to surviving stressful conditions is the accumulation of ureides. Ureides are recognized as important nitrogen-rich compounds involved in recycling nitrogen in plants to support growth and reproduction. Amongst them, allantoin not only serves as a transportable nitrogen-rich compound, but has also been suggested to protect plants from abiotic stresses via minimizing oxidative damage. This work focuses on the effect of cadmium (Cd) on ureide metabolism in Arabidopsis, in order to clarify the potential role of allantoin in plant tolerance to heavy metals. In response to Cd treatment, allantoin levels increase in Arabidopsis thaliana, ecotype Col-0, due to reduced allantoinase (ALN) gene expression and enzyme activity. This coincides with increases in uricase (UO) transcripts. UO and ALN encode the enzymes for the production and degradation of allantoin, respectively. ALN-negative aln-3 Arabidopsis mutants with elevated allantoin levels demonstrate resistance to soil-applied CdCl 2 , up to 1,500 μM. Although aln-3 mutants take up and store more Cd within their leaf tissue, they contain less damaging superoxide radicals. The protective mechanism of aln-3 mutants appears to involve enhancing the activity of antioxidant enzymes such as superoxide dismutase and ascorbate peroxidase., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Ureides are nitrogenous compounds derived from purine catabolism which contribute to nitrogen recycling in plants. Accumulation of ureide compounds has been reported in a number of plant species under stress conditions, suggesting their involvement in plants' response to stress. In this research a biochemical and molecular approach was applied to address the ureide accumulation under abiotic stress conditions in Arabidopsis thaliana. Ureide concentration and changes in expression of ureide metabolic genes were examined in response to drought, NaCl and mannitol treatments. Additionally, an Arabidopsis allantoinase (ALN) mutant with constitutive accumulation of a ureide compound, allantoin, was used to investigate the impact of high levels of this compound on drought and NaCl stress responses. In the leaf tissue of adult plants allantoin accumulated in response to soil drying. Transcription of urate oxidase (UO), involved in allantoin production, was highly up-regulated under the same conditions. Allantoin and allantoate also accumulated in seedlings following treatment with NaCl or mannitol. aln mutants with enhanced levels of allantoin exhibited higher tolerance to drought and NaCl. Hydrogen peroxide and superoxide did not accumulate in the aln mutant leaves to the same degree in response to drought when compared to the wild-type. Our results suggest that ureide metabolism and accumulation contribute to the abiotic stress response which is regulated, at least in part, at the transcriptional level. Higher concentrations of allantoin in the mutant elevates abiotic stress tolerance, possibly by reducing oxidative damage., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

With the sequencing of genomes from many organisms now complete and the development of high-throughput sequencing, life science research has entered the functional post-genome era. Therefore, deciphering the function of genes and how they interact is in greater demand. To study an unknown gene, the basic methods are either overexpression or gene knockout by creating transgenic plants, and gene construction is usually the first step. Although traditional cloning techniques using restriction enzymes or a site-specific recombination system (Gateway or Clontech cloning technology) are highly useful for efficiently transferring DNA fragments into destination plasmids, the process is time consuming and expensive. To facilitate the procedure of gene construction, we designed a TA-based cloning system in which only one step was needed to subclone a DNA fragment into vectors. Such a cloning system was developed from the pGreen binary vector, which has a minimal size and facilitates construction manipulation, combined with the negative selection marker gene ccdB, which has the advantages of eliminating the self-ligation background and directly enabling high-efficiency TA cloning technology. We previously developed a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, subcellular localization analysis and promoter activity detection. Our results show that such a system is highly efficient and serves as a high-throughput platform for transient or stable transformation in plants for functional genome research.

Mutation of either arginase structural gene (ARGAH1 or ARGAH2 encoding arginine [Arg] amidohydrolase-1 and -2, respectively) resulted in increased formation of lateral and adventitious roots in Arabidopsis (Arabidopsis thaliana) seedlings and increased nitric oxide (NO) accumulation and efflux, detected by the fluorogenic traps 3-amino,4-aminomethyl-2',7'-difluorofluorescein diacetate and diamino-rhodamine-4M, respectively. Upon seedling exposure to the synthetic auxin naphthaleneacetic acid, NO accumulation was differentially enhanced in argah1-1 and argah2-1 compared with the wild type. In all genotypes, much 3-amino,4-aminomethyl-2',7'-difluorofluorescein diacetate fluorescence originated from mitochondria. The arginases are both localized to the mitochondrial matrix and closely related. However, their expression levels and patterns differ: ARGAH1 encoded the minor activity, and ARGAH1-driven beta-glucuronidase (GUS) was expressed throughout the seedling; the ARGAH2::GUS expression pattern was more localized. Naphthaleneacetic acid increased seedling lateral root numbers (total lateral roots per primary root) in the mutants to twice the number in the wild type, consistent with increased internal NO leading to enhanced auxin signaling in roots. In agreement, argah1-1 and argah2-1 showed increased expression of the auxin-responsive reporter DR5::GUS in root tips, emerging lateral roots, and hypocotyls. We propose that Arg, or an Arg derivative, is a potential NO source and that reduced arginase activity in the mutants results in greater conversion of Arg to NO, thereby potentiating auxin action in roots. This model is supported by supplemental Arg induction of adventitious roots and increased NO accumulation in argah1-1 and argah2-1 versus the wild type.

The detailed expression patterns of transcripts of two Arabidopsis arginase genes, ARGAH1 and ARGAH2, have not been previously described, and phylogenetic analysis suggests that they diverged independently of duplication events in other lineages. Therefore, we used beta-glucuronidase reporter fusions and quantitative reverse-transcriptase PCR to analyze tissue-specific expression of ARGAH1 and ARGAH2 during Arabidopsis development, and in response to the availability of nutrients and exposure to methyl jasmonate (MeJA). We demonstrated tissue-specific transcript expression and enzyme activity in pollen for ARGAH1, but not ARGAH2. Conversely, we demonstrated MeJA-inducibility of ARGAH2, but not ARGAH1. In addition, we used microarrays to identify genes for which transcript abundance following MeJA treatment differed in wild type and ARGAH2 mutants. These ARGAH2 and MeJA responsive genes included a putative pathogenesis-related protein pathogenesis response-1 (At2g14610), and a gene of unknown function (At5g03090). Interestingly, these genes had opposite responses to the loss of ARGAH2, suggesting multiple downstream effects of arginase activity, following MeJA treatment. These results, and the variety and complexity of expression patterns of ARGAH1 and ARGAH2 transcript expression and their related reporter gene fusions that we observed point to multiple functions of arginase genes in Arabidopsis, some of which have resulted through a sub-functionalization not shared by all angiosperms.

To shed light on the metabolic role of two mitochondrial transporters for basic amino acids in Arabidopsis, we compared their functional properties in liposomes and expression during germination. Recombinant and purified BAC2, as previously reported for BAC1, transported various basic L-amino acids upon reconstitution in phospholipid vesicles. Both displayed highest affinity for arginine with similar Km and Vmax. However, BAC2 transported citrulline for which BAC1 had little or no affinity. Furthermore, BAC2 was less stereospecific than BAC1, transporting D-arginine and D-lysine at significant rates, and displayed a striking alkaline pH optimum (pH 8.0) whereas BAC1 activity was unaltered from pH 7.0 to 9.0. By semi-quantitative RT-PCR BAC1 transcript levels were found to be higher than those of BAC2 in germinated seeds. However, BAC2 expression transiently increased 2 days after germination. Disruption of the Arabidopsis arginase structural genes (ARGAH1 or ARGAH2) accentuated the increases of transcript levels of BAC1 at germination and of BAC2 2 days after germination and from 6 days on. Early expression of BAC1 and BAC2 is consistent with the delivery of arginine, released from seed reserves, to mitochondrial arginase and the export of ornithine. Increase of BAC2 transcript levels later in seedling development is consistent with roles in NO, polyamine or proline metabolism--processes involving arginine, citrulline and/or ornithine.

We report the identification and cloning of an allantoate amidohydrolase (allantoate deiminase, EC 3.5.3.9) cDNA from Arabidopsis thaliana (L.) Heynh. This sequence, which we term Arabidopsis thaliana Allantoate Amidohydrolase (AtAAH), was shown to be functional by complementation of Saccharomyces cerevisiae dal2 mutants, blocked in allantoate degradation. Following transfer to a medium containing allantoin as the sole nitrogen source, Ataah T-DNA insertion mutants were severely impaired and eventually died. Ataah mutants demonstrated higher allantoate levels than wild-type plants in the presence and absence of exogenous ureides, supporting a block in allantoate catabolism. AtAAH transcript was detected in all tissues examined by RT-PCR, consistent with a function in purine turnover in Arabidopsis. To our knowledge this is the first allantoate amidohydrolase gene identified in any plant species.

In Arabidopsis thaliana (L.) Heynh., AtPhr2 and AtNsr1 encode proteins with MYB-like and alpha-helical domains. They resemble CrPsr1, a nuclear-localized MYB protein that is critical for acclimation to phosphorous (P) starvation in the alga Chlamydomonas reinhardtii. Reverse transcription-polymerase chain reaction analysis of the first unique exons indicated that AtPhr2 mRNA increased as early as 6 h after P deprivation (-P), whereas nitrogen deprivation (-N) had no effect. The AtNsr1 mRNA level increased exclusively under -N, an increase first noted by 2 days in -N. In spite of P- and N-specific effects on expression of AtPhr2 and AtNsr1 there appeared to be P-N cross-talk at the whole-plant level. Total non-secreted acid phosphatase activity increased under both -P and -N within 2 days of deprivation. Further, the pho2-1/pho2-1 mutant, reported to be a phosphate accumulator, showed no increase in AtPhr2 mRNA in response to -P and a 70% reduction in the response of AtNsr1 mRNA to -N. Consistent with this pattern, there was no increase in acid phosphatase activity in pho2-1/pho2-1 plants deprived of P or N. However, when deprived of P, pho2-1/pho2-1 plants accumulated much higher levels of nitrate. T-DNA disruption of AtNsr1 resulted in altered expression of at least one nitrate transporter (AtNRT2.5). Further evidence of cross-talk between N and P responses was altered expression of N-responsive genes in pho2-1/pho2-1.