Joseph C. Polacco

Essential Role of Urease in Germination of Nitrogen-Limited Arabidopsis thaliana Seeds

In Arabidopsis thaliana, urease transcript levels increased sharply between 2 and 4 d after germination (DAG) and were maintained at maximal levels until at least 8 DAG. Seed urease specific activity declined upon germination but began to increase in seedlings 2 DAG, reaching approximately 75% of seed activity by 8 DAG. Urea levels showed a small transient increase 1 DAG and then approximately paralleled urease activity, reaching maximal levels at approximately 9 DAG. Urease inhibition with phenylphosphorodiamidate resulted in a 2- to 4-fold increase in urea levels throughout seedling development. Arginine pools (0–8 DAG) changed approximately in parallel with the urea pool. Consistent with arginine being a major source of urea, arginase activity increased 10-fold in the interval 0 to 6 DAG. Allopurinol, a xanthine dehydrogenase inhibitor, had no effect on urea levels up to 3 DAG but reduced the urea pool by 30 to 40% during the interval 5 to 8 DAG, suggesting that purine degradation contributed to the urea pool well after germination, if at all. In aged Arabidopsis seeds, there was a correlation between phenylphosphorodiamidate inactivation of urease and germination inhibition, the latter overcome by NH4NO3 or amino acids. Since urease activity, urea precursor, and urea increase in young seedlings, and since urease inactivation results in a nitrogen-reversible inhibition of germination, we propose that urease recycles urea-nitrogen in the seedling.

Nitric oxide functions as a positive regulator of root hair development.

The root epidermis is composed by two cell types: trichoblasts (or hair cells) and atrichoblasts (or non-hair cells). In lettuce (Lactuca sativa cv. Grand Rapids var. Rapidmor oscura) plants grown hydroponically in water, the root epidermis did not form root hairs. The addition of 10 µM sodium nitroprusside (SNP), a nitric oxide (NO) donor, resulted in almost all rhizodermal cells differentiated into root hairs. Treatment with the synthetic auxin 1-naphthyl acetic acid (NAA) displayed a significant increase of root hair formation (RHF) that was prevented by the specific NO scavenger carboxy-PTIO (cPTIO). In Arabidopsis, two mutants have been shown to be defective in NO production and to display altered phenotypes in which NO is implicated. Arabidopsis nos1 has a mutation in an NO synthase structural gene (NOS1), and the nia1 nia2 double mutant is null for nitrate reductase (NR) activity. We observed that both mutants were affected in their capacity of developing root hairs. Root hair elongation was significantly reduced in nos1 and nia1 nia2 mutants as well as in cPTIO-treated wild type plants. A correlation was found between endogenous NO level in roots detected by the fluorescent probe DAF-FM DA and RHF. In Arabidopsis, as well as in lettuce, cPTIO blocked the NAA-induced root hair elongation. Taken together, these results indicate that: (i) NO is a critical molecule in the process leading to RHF, and (ii) NO is involved in the auxin-signaling cascade leading to RHF.

Roles of Urease in Plant Cells

Publisher Summary This chapter discusses the role of urease in plant cells. Urease is important for efficient nitrogen assimilation. Considerable amounts of plant nitrogen flow through urea (urease substrate), which can be recycled only by urease action. This recapture can have significant quality impact on protein rich crops. It appears to have an important role in germination of protein poor seeds. The urease substrate urea is derived from arginine and ureides. Arginine is the richest nitrogen repository among the amino acids of seed storage proteins. On the other hand, ureides are not only significant sources of nitrogen in nucleic acid turnover but are also a predominant transport from of fixed nitrogen in soybean and other tropical legumes. Urease-negative plants accumulate substantial, nonutilizable urea in both maternal and embryonic tissue. During germination of urease-negative seeds, further urea accumulates as a dead end in nitrogen metabolism. Abundant seed ureases, such as, Sumners jackbean urease, may play a chemical defense role. All of the ureases, both from bacteria and plants, resemble each other in primary structure and in their requirement for accessory genes.

Significance of nickel for plant growth and metabolism

Ni is the most recent candidate to be added to the list of 13 essential mineral elements for higher plants although failure to complete the life cycle in the absence of Ni has only been demonstrated in a few plant species. Ni is considered an essential element primarily because of its function as an irreplaceable component of urease which is responsible for the hydrolysis of urea N, and which seems to be the only proven nutritional function of Ni in higher plants. For production of full urease activity and growth on urea N a critical deficiency level of around 100 μg kg—1 DW seems appropriate, while plants depending on mineral N may have a lower Ni requirement. Ni has also other effects on plant growth, of which the phytosanitary action is possibly most significant in the field. The incorporation of Ni into urease apoprotein requires the active participation of several accessory proteins, and mutations in genes coding the accessory proteins as well as the urease apoprotein have been exploited to characterise aspects of urease activation. The mobility of Ni within the plant, as compared to other heavy metals, is usually high, although little is known of the uptake mechanisms and the form of transported Ni under Ni-deprived conditions. This as well as other effects of Ni that cannot be related to its structural component of urease, remain to be elucidated. Die Bedeutung von Nickel fur das Wachstum und den Stoffwechsel von Pflanzen Ni ist der jungste Kandidat fur die Liste der bisher 13 essentiellen mineralischen Nahrelemente hoherer Pflanzen, obwohl der Beweis fur seine Notwendigkeit zur Vollendung des Lebenszyklus einer Pflanze erst bei wenigen Arten gelang. Die Essentialitat von Ni begrundet sich insbesondere auf dessen Funktion als unersetzlicher Bestandteil des Enzyms Urease, welches die Hydrolyse von Harnstoff katalysiert. Dies ist die bislang einzige nachgewiesene Funktion von Ni in der hoheren Pflanze. Hinsichtlich des Wachstums harnstoffernahrter Pflanzen und der Ureaseaktivitat konnte ein kritischer Gehalt von etwa 100 μg kg—1 TM abgeleitet werden, wahrend Pflanzen, die mineralischen N erhalten, einen wesentlich geringeren Ni-Bedarf haben. Ni hat noch weitere Effekte auf das Pflanzenwachstum, von denen die phytosanitaren Wirkungen unter Feldbedingungen sicherlich die wichtigsten sind. Der Einbau von Ni in die Ureaseapoproteine erfordert die aktive Beteiligung mehrerer akzessorischer Proteine und Mutationen der Gene, die diese akzessorischen Proteine und die Ureasestrukturgene kodieren, wurden erfolgreich zur Aufklarung zahlreicher Aspekte der Ureaseaktivierung eingesetzt. Die innerpflanzliche Mobilitat von Ni ist, verglichen mit anderen Schwermetallen, normalerweise relativ hoch, doch liegen nur wenige Informationen uber die Ni-Aufnahme und transportierten Ni-Spezies bei niedrigem Ni-Angebot vor. Diese und andere Aspekte der Ni-Ernahrung, die nicht auf dessen strukturelle Bedeutung in der Urease zuruckgefuhrt werden konnen, harren ihrer Untersuchung.

Arginase-Negative Mutants of Arabidopsis Exhibit Increased Nitric Oxide Signaling in Root Development

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 β-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.

Refined glufosinate selection in Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill].

Modern genetic analysis and manipulation of soybean (Glycine max) depend heavily on an efficient and dependable transformation process, especially in public genotypes from which expressed sequence tag (EST), bacterial artificial chromosome and microarray data have been derived. Williams 82 is the subject of EST and functional genomics analyses. However, it has not previously been transformed successfully using either somatic embryogenesis-based or cotyledonary-node transformation methods, the two predominant soybean transformation systems. An advance has recently been made in using antioxidants to enhance Agrobacterium infection of soybean. Nonetheless, an undesirable effect of using these antioxidants is the compromised recovery of transgenic soybean when combined with the use of the herbicide glufosinate as a selective agent. Therefore, we optimized both Agrobacterium infection and glufosinate selection in the presence of l-cysteine for Williams 82. We have recovered transgenic lines of this genotype with an enhanced transformation efficiency using this herbicide selection system.

Nitric oxide in plant growth, development and stress physiology

Higher Plant Mitochondria as a Source for NO.- Nitric Oxide - A Product of Plant Nitrogen Metabolism.- NO-Based Signaling in Plants.- S-Nitrosylation in Plants - Spectrum and Selectivity.- Enzymatic Sources of Nitric Oxide during Seed Germination.- Seeking the Role of NO in Breaking Seed Dormancy.- Nitric Oxide Functions as Intermediate in Auxin, Abscisic Acid, and Lipid Signaling Pathways.- Nitric Oxide in Cytokinin and Polyamine Signaling: Similarities and Potential Crosstalk.- Nitric Oxide and Plant Ion Channel Control.- Nitric Oxide in Nitrogen-Fixing Symbiosis.- Nitrosative Stress in Plants: A New Approach to Understand the Role of NO in Abiotic Stress.- Nitric Oxide-Mediated Signaling Functions During the Plant Hypersensitive Response.- Nitric Oxide in Cell-to-Cell Communication Coordinating the Plant Hypersensitive Response.- Mitochondrial Nitric Oxide Synthesis During Plant-Pathogen Interactions: Role of Nitrate Reductase in Providing Substrates.- Nitric Oxide as an Alternative Electron Carrier During Oxygen Deprivation.- Fluorometric Detection of Nitric Oxide with Diaminofluoresceins (DAFs): Applications and Limitations for Plant NO Research.

Arginine degradation by arginase in mitochondria of soybean seedling cotyledons.

Abstract. Arginase (EC 3.5.3.1) localization was studied in soybean (Glycine max L.) seedling cotyledons. Subcellular fractionation in a discontinuous Percoll gradient showed that arginase was localized in the mitochondrion. Arginine (Arg) uptake by mitochondria was demonstrated by co-sedimentation of [3H]Arg-derived label and the mitochondrial marker enzyme cytochrome c oxidase. Arginine uptake was complete in about 10 min. Since detergent but not NaCl released most label, we conclude that Arg was taken up and not bound to the organellar surface. Arginine transport was not saturable, at least up to 20 mM. Basic amino acids were the best inhibitors of Arg uptake. The uncoupler 2,4-dinitrophenol did not inhibit Arg uptake. At least 30% of l-[guanido-14C]Arg taken up by mitochondria was degraded by arginase in seedling cotyledons, while little or no degradation was detected in mitochondria from developing embryos, even though the Arg uptake level was similar in both mitochondrial preparations. These results are consistent with our previously reported pattern of arginase expression and urea accumulation during embryo development and seed germination (A. Goldraij and J.C. Polacco, 1999, Plant Physiol. 119: 297–303). The lack of Arg degradation allows developing embryos to conserve Arg, the main N-reserve amino acid utilized by germinating soybean.

Quantitative Conversion of Phytate to Inorganic Phosphorus in Soybean Seeds Expressing a Bacterial Phytase

Phytic acid (PA) contains the major portion of the phosphorus in the soybean (Glycine max) seed and chelates divalent cations. During germination, both minerals and phosphate are released upon phytase-catalyzed degradation of PA. We generated a soybean line (CAPPA) in which an Escherichia coli periplasmic phytase, the product of the appA gene, was expressed in the cytoplasm of developing cotyledons. CAPPA exhibited high levels of phytase expression, ≥90% reduction in seed PA, and concomitant increases in total free phosphate. These traits were stable, and, although resulted in a trend for reduced emergence and a statistically significant reduction in germination rates, had no effect on the number of seeds per plant or seed weight. Because phytate is not digested by monogastric animals, untreated soymeal does not provide monogastrics with sufficient phosphorus and minerals, and PA in the waste stream leads to phosphorus runoff. The expression of a cytoplasmic phytase in the CAPPA line therefore improves phosphorus availability and surpasses gains achieved by other reported transgenic and mutational strategies by combining in seeds both high phytase expression and significant increases in available phosphorus. Thus, in addition to its value as a high-phosphate meal source, soymeal from CAPPA could be used to convert PA of admixed meals, such as cornmeal, directly to utilizable inorganic phosphorus.

Urease Is Not Essential for Ureide Degradation in Soybean

The hypothesis that soybean (Glycine max L. [Merrill]) catabolizes ureides to urea to a physiologically significant extent was tested and rejected. Urease-negative (eu3-e1/eu3-e1) plants were supported by fixed N2 or by 2 mM NH4NO3, so that xylem-borne nitrogen contained predominantly ureides (allantoin and allantoic acid) or amide amino acids, respectively. Seed nitrogen yield was equal on either nitrogen regime, although 35-d-old fixing plants accumulated about 6 times more leaf urea. In callus, lack of an active urease reduced growth on either arginine or allantoin as the sole nitrogen source, but the reduction was greater on arginine (73%) than on allantoin (39%). Furthermore, urease-negative cells accumulated 17 times more urea than urease-positive cells on arginine; for allantoin the ratio was 1.8. Urease-negative callus accumulated urea at 3% the rate of seedlings. To test whether urea accumulating in urease-negative seedlings was derived from ureides, seeds were first allowed to imbibe in 1 mM allopurinol, an inhibitor of ureide formation. Seedling ureides were decreased by 90%, but urea levels were unchanged. Thus, ureides are poor precursors of urea, which was confirmed in seedlings that converted no more than 5% of seed-absorbed [14C-ureido]allantoate to [14C]urea, whereas 40 to 70% of [14C-guanido]arginine was recovered as [14C]urea.