Comparative skeletal anatomy of salt marsh and western harvest mice in relation to locomotor ecology.

The salt marsh harvest mouse (Reithrodontomys raviventris) is an endangered species, endemic to the San Francisco Bay Estuary, that co-occurs with the more broadly distributed species, the western harvest mouse (Reithrodontomys megalotis). Despite their considerable external morphological similarities, the northern subspecies of salt marsh harvest mice have relatively longer and thicker tails than do western harvest mice, which may be related to their abilities to climb emergent marsh vegetation to avoid tidal inundation. We used micro-CT to compare post-cranial skeletal anatomy between the salt marsh and western harvest mouse, to examine whether the salt marsh harvest mouse's restriction to brackish marshes is associated with skeletal adaptations for scansorial locomotion. We found that salt marsh harvest mice exhibited a deeper 3rd caudal vertebra, a more caudally located longest tail vertebra, craniocaudally longer tail vertebrae, and a longer digit III proximal phalanx than western harvest mice. These phalangeal and vertebral characteristics are known to decrease body rotations during climbing, increase contact with substrates, and decrease fall susceptibility in arboreal mammals, suggesting that the salt marsh harvest mouse may be morphologically specialized for scansorial locomotion, adaptive for its dynamic wetland environment.

The benefits of woody plant stem photosynthesis extend to hydraulic function and drought survival in Parkinsonia florida.

As climate change exacerbates drought stress in many parts of the world, understanding plant physiological mechanisms for drought survival is critical to predicting ecosystem responses. Stem net photosynthesis, which is common in arid environments, may be a drought survival trait, but whether the additional carbon fixed by stems contributes to plant hydraulic function and drought survival in arid land plants is untested. We conducted a stem light-exclusion experiment on saplings of a widespread North American desert tree species, Parkinsonia florida L., and after shading acclimation, we then subjected half of the plants to a drought treatment to test the interaction between light exclusion and water limitation on growth, leaf and stem photosynthetic gas exchange, xylem embolism assessed with micro-computed tomography and gravimetric techniques, and survival. Growth, stem photosynthetic gas exchange, hydraulic function and survival all showed expected reductions in response to light exclusion. However, stem photosynthesis mitigated the drought-induced reductions in gas exchange, xylem embolism (percent loss of conductivity, PLC) and mortality. The highest mortality was in the combined light exclusion and drought treatment, and was related to stem PLC and native sapwood-specific hydraulic conductivity. This research highlights the integration of carbon economy and water transport. Our results show that additional carbon income by photosynthetic stems has an important role in the growth and survival of a widespread desert tree species during drought. This shift in function under conditions of increasing stress underscores the importance of considering stem photosynthesis for predicting drought-induced mortality not only for the additional supply of carbon, but also for its extended benefits for hydraulic function.

Drought resistance in two populations of invasive Tamarix compared using multiple methods.

An on-going question in plant hydraulic research is whether there is intra-specific variability and/or plasticity in xylem traits. Plasticity could be important in taxa that colonize diverse habitats. We used Tamarix, a non-native woody plant, to investigate population differences in hydraulic conductivity (Ks), vulnerability-to-embolism curves and vessel anatomy. We also conducted a season-long drought experiment to determine water potentials associated with crown dieback of field-grown plants. We measured vessel length and diameter, and compared visual (micro-computed tomography; microCT) and hydraulic methods to quantify percentage loss in hydraulic conductivity (PLC). Among plants grown in a common environment, we did not find differences in our measured traits between two populations of Tamarix that differ in salinity at their source habitats. This taxon is relatively vulnerable to embolism. Within samples, large diameter vessels displayed increased vulnerability to embolism. We found that the microCT method overestimated theoretical conductivity and underestimated PLC compared with the hydraulic method. We found agreement for water potentials leading to crown dieback and results from the hydraulic method. Saplings, grown under common conditions in the present study, did not differ in their xylem traits, but prior research has found difference among source-site grown adults. This suggests that plasticity may be key in the success of Tamarix occurring across a range of habits in the arid southwest USA.

Xylem structure and hydraulic function in roots and stems of chaparral shrub species from high and low elevation in the Sierra Nevada, California.

Xylem structure and hydraulics were compared between individuals at lower and upper elevation distribution limits for five chaparral shrub species along a steep transect in the southern Sierra Nevada, California, USA. Higher-elevation plants experienced frequent winter freeze-thaw events and increased precipitation. We hypothesized that environmental differences would lead to xylem trait differences between high and low elevations, but predictions were complicated because both water stress (low elevation) and freeze-thaw events (high elevation) may select for similar traits, such as narrow vessel diameter. We found significant changes in the ratio of stem xylem area to leaf area (Huber value) between elevations, with more xylem area required to support leaves at low elevations. Co-occurring species significantly differed in their xylem traits, suggesting diverse strategies to cope with the highly seasonal environment of this Mediterranean-type climate region. Roots were more hydraulically efficient and more vulnerable to embolism relative to stems, potentially due to roots being buffered from freeze-thaw stress, which allows them to maintain wider diameter vessels. Knowledge of the structure and function of both roots and stems is likely important in understanding whole-plant response to environmental gradients.

Functional response and resistance to drought in seedlings of six shrub species with contrasting leaf traits from the Mediterranean Basin and California.

Extreme drought events during post-fire regeneration are becoming increasingly frequent in Mediterranean-type ecosystems. Understanding how plants with different traits and origins respond to such conditions during early life stages is therefore critical for assessing the effect of climate change. Here, seedlings of three Cistus (semi-deciduous malacophylls from the Mediterranean Basin) and three Ceanothus (evergreen sclerophylls from California) species, two post-fire seeder genera with contrasting leaf traits, were subjected to complete water deprivation for 3 months in a common garden experiment. The leaf and plant structure and plant tissue water relations were characterized before the drought, and the functional responses (water availability, gas exchange and fluorescence) were monitored during the drought. Both genera exhibited contrasting leaf structure and tissue water relations traits, with higher leaf area and specific leaf area as well as higher osmotic potential at maximum turgor and turgor loss point in Cistus than Ceanothus. During drought, Ceanothus showed a more conservative use of water than Cistus, with a water potential less sensitive to decreasing soil moisture and a strong decline in photosynthesis and stomatal conductance in response to water deficit, but also a level of fluorescence more responsive to drought than Cistus. However, we could not find a different degree of drought resistance between the genera. This was particularly clear between Cistus ladanifer L. and Ceanothus pauciflorus DC., the two most functionally contrasting species, but at the same time, the two most drought-resistant. Our findings demonstrate that species with different leaf traits and functional responses to water stress may not differ in their degree of drought resistance, at least during the seedling stage. This underlines the need to take general categorizations by genus or functional types with caution and to deepen our knowledge about the Mediterranean-type species ecophysiology, especially during early life stages, in order to anticipate their vulnerability to climate change.

Seasonal patterns of increases in stem girth, vessel development, and hydraulic function in deciduous tree species.

BACKGROUND AND AIMS: The onset of spring growth and vessel formation were examined within three deciduous woody plant species, Acer rubrum, Populus balsamifera ssp. trichocarpa and Quercus rubra. We were broadly interested in the lag between the onset of girth expansion and the formation of mature and hydraulically conductive vessels within the new xylem. METHODS: Dendrometers were installed on 20 trees (6-7 per species), and expansion of both bole and distal stems was monitored throughout the growing season in a common garden. For each species, four to six distal stems were harvested every other week for anatomical examination of vessel formation. Additionally, for Populus and Quercus, hydraulic conductivity measurements and active xylem staining were completed on all stem samples. KEY RESULTS: For all three species, the timing of girth expansion was similar. Expansion of distal branches occurred 12-37 d earlier than that of the bole. Vessel formation initiated several weeks prior to leaf-out, but no new earlywood vessels were mature at the time of bud break for Acer and Populus and only a few were present in Quercus. Initial stem girth expansion occurred 2 to >6 weeks before the maturation of the first current-year vessels, and there was an additional delay of up to 4 weeks before mature vessels became hydraulically functional. Hydraulic conductivity was strongly correlated with the number and diameter of stained vessels. CONCLUSIONS: Bud break and leaf expansion relied predominantly on water supplied by vessels formed during prior seasons. Early-season activity is likely affected by the function of older xylem vessels and the environmental factors that influence their structure and function. Understanding the functional lifespan of vessels and the varying contributions of new and older vessels to conductivity are critical to understanding of the phenology and vascular function of long-lived woody plants in response to changing climates.

Vegetation-type conversion of evergreen chaparral shrublands to savannahs dominated by exotic annual herbs: causes and consequences for ecosystem function.

Woody, evergreen shrublands are the archetypal community in mediterranean-type ecosystems, and these communities are profoundly changed when they undergo vegetation-type conversion (VTC) to become annual, herb-dominated communities. Recently, VTC has occurred throughout southern California chaparral shrublands, likely with changes in important ecosystem functions. The mechanisms that lead to VTC and subsequent changes to ecosystem processes are important to understand as they have regional and global implications for ecosystem services, climate change, land management, and policy. The main drivers of VTC are altered fire regimes, aridity, and anthropogenic disturbance. Some changes to ecosystem function are certain to occur with VTC, but their magnitudes are unclear, whereas other changes are unpredictable. I present two hypotheses: (1) VTC leads to warming that creates a positive feedback promoting additional VTC, and (2) altered nitrogen dynamics create negative feedbacks and promote an alternative stable state in which communities are dominated by herbs. The patterns described for California are mostly relevant to the other mediterranean-type shrublands of the globe, which are biodiversity hotspots and threatened by VTC. This review examines the extent and causes of VTC, ecosystem effects, and future research priorities.

Trade-offs among transport, support, and storage in xylem from shrubs in a semiarid chaparral environment tested with structural equation modeling.

The xylem in plants is specialized to transport water, mechanically support the plant body, and store water and carbohydrates. Balancing these functions leads to trade-offs that are linked to xylem structure. We proposed a multivariate hypothesis regarding the main xylem functions and tested it using structural equation modeling. We sampled 29 native shrub species from field sites in semiarid Southern California. We quantified xylem water transport (embolism resistance and transport efficiency), mechanical strength, storage of water (capacitance) and starch, minimum hydrostatic pressures (Pmin), and proportions of fibers, vessels, and parenchyma, which were treated as a latent variable representing "cellular trade-offs." We found that xylem functions (transport, mechanical support, water storage, and starch storage) were independent, a result driven by PminPmin was strongly and directly or indirectly associated with all xylem functions as a hub trait. More negative Pmin was associated with increased embolism resistance and tissue strength and reduced capacitance and starch storage. We found strong support for a trade-off between embolism resistance and transport efficiency. Tissue strength was not directly associated with embolism resistance or transport efficiency, and any associations were indirect involving Pmin With Pmin removed from the model, cellular trade-offs were central and related to all other traits. We conclude that xylem traits are broadly governed by functional trade-offs and that the Pmin experienced by plants in the field exerts a strong influence over these relationships. Angiosperm xylem contains different cell types that contribute to different functions and that underpin trade-offs.

Xylem biomechanics, water storage, and density within roots and shoots of an angiosperm tree species.

Xylem is a complex tissue that forms the bulk of tree bodies and has several functions, including to conduct water, store water and nutrients, and biomechanically support the plant body. We examined how xylem functional traits varied at different positions within 9-year-old Populus balsamifera subsp. trichocarpa. Whole trees were excavated, and xylem samples were collected at 1-m increments along the main root-to-shoot axis of six trees, from root tip to shoot tip. We examined biomechanical and water-storage traits of the xylem, including using a non-invasive imaging technique to examine water content within long, intact branches (high-resolution computed tomography; microCT). Xylem density, strength, and stiffness were greater in shoots than roots. Along the main root-to-shoot axis, xylem strength and stiffness were greatest at shoot tips, and the tissue became linearly weaker and less stiff down the plant and through the root. Roots had greater water storage with lower biomechanical support, and shoots had biomechanically stronger and stiffer xylem with lower water storage. These findings support trade-offs among xylem functions between roots and shoots. Understanding how xylem functions differ throughout tree bodies is important in understanding whole-tree functioning and how terrestrial plants endure numerous environmental challenges over decades of growth.

Detection of an enzyme isomechanism by means of the kinetics of covalent inhibition.

Turnover of substrates by many enzymes involves free enzyme forms that differ from the stable form of the enzyme in the absence of substrate. These enzyme species, known as isoforms, have, in general, different physical and chemical properties than the native enzymes. They usually occur only in small concentrations under steady state turnover conditions and thus are difficult to detect. We show in this paper that in one particular case of an enzyme (a class C ß-lactamase) with specific substrates (cephalosporins) the presence of an enzyme isoform (E') can be detected by means of its different reactivity than the native enzyme (E) with a class of covalent inhibitors (phosphonate monoesters). Generation of E' from E arises either directly from substrate turnover or by way of a branched path from an acyl-enzyme intermediate. The relatively slow spontaneous restoration of E from E' is accelerated by certain small molecules in solution, for example cyclic amines such as imidazole and salts such as sodium chloride. Solvent deuterium kinetic isotope effects and the effect of methanol on cephalosporin turnover showed that for both E and E', kcat is limited by deacylation of an acyl-enzyme intermediate rather than by enzyme isomerization.

Starch storage capacity of sapwood is related to dehydration avoidance during drought.

PREMISE: The xylem tissue of plants performs three principal functions: transport of water, support of the plant body, and nutrient storage. Tradeoffs may arise because different structural requirements are associated with different functions or because suites of traits are under selection that relate to resource acquisition, use, and turnover. The structural and functional basis of xylem storage is not well established. We hypothesized that greater starch storage would be associated with greater sapwood parenchyma and reduced fibers, which would compromise resistance to xylem tensions during dehydration. METHODS: We measured cavitation resistance, minimum water potential, starch content, and sapwood parenchyma and fiber area in 30 species of southern California chaparral shrubs (evergreen and deciduous). RESULTS: We found that species storing greater starch within their xylem tended to avoid dehydration and were less cavitation resistant, and this was supported by phylogenetic independent contrasts. Greater sapwood starch was associated with greater parenchyma area and reduced fiber area. For species without living fibers, the associations with parenchyma were stronger, suggesting that living fibers may expand starch storage capacity while also contributing to the support function of the vascular tissue. Drought-deciduous species were associated with greater dehydration avoidance than evergreens. CONCLUSIONS: Evolutionary forces have led to an association between starch storage and dehydration resistance as part of an adaptive suite of traits. We found evidence for a tradeoff between tissue mechanical traits and starch storage; moreover, the evolution of novel strategies, such as starch-storing living fibers, may mitigate the strength of this tradeoff.

Factors controlling drought resistance in grapevine (Vitis vinifera, chardonnay): application of a new microCT method to assess functional embolism resistance.

PREMISE: Quantifying resistance to embolism in woody plants is important for understanding their drought response. Methods to accurately quantify resistance to embolism continue to be debated. METHODS: We used a new microCT-based approach that quantifies embolized conduits and also analyzes conductive conduits by using an x-ray-dense, iodine-rich tracer that moves though the vascular system and can easily be observed in microCT images. Many previous microCT studies assumed that all conduits were initially conductive, which may not be the case if there are developing or occluded conduits. We compared microCT results to a standard benchtop dehydration method and a centrifuge method. During dehydration, we measured gas exchange and quantified water potential at mortality. RESULTS: Our microCT curves agreed with previously published microCT curves from the same greenhouse-grown cultivar. We found a significant difference in embolism estimates if we assumed that all water-filled conduits were functional rather than only those containing tracer. Embolism estimates from microCT differed from both the benchtop and centrifuge methods. The benchtop and centrifuge methods did not differ from one another. CONCLUSIONS: The new microCT method presented here is valuable in sampling species that may contain nonconductive conduits. Disagreement between microCT and two other methods was likely due to differences in the ways they quantify embolism. MicroCT assess the theoretical effect of embolism, whereas benchtop and centrifuge methods directly measure hydraulic conductivity. The theoretical approach does not fully account for the resistances of flow through a complex 3D vascular network.

Elusive structural changes of New Delhi metallo-ß-lactamase revealed by ultraviolet photodissociation mass spectrometry.

We use mass spectrometry (MS), under denaturing and non-denaturing solution conditions, along with ultraviolet photodissociation (UVPD) to characterize structural variations in New Delhi metallo-ß-lactamase (NDM) upon perturbation by ligands or mutation. Mapping changes in the abundances and distributions of fragment ions enables sensitive detection of structural alterations throughout the protein. Binding of three covalent inhibitors was characterized: a pentafluorphenyl ester, an O-aryloxycarbonyl hydroxamate, and ebselen. The first two inhibitors modify Lys211 and maintain dizinc binding, although the pentafluorophenyl ester is not selective (Lys214 and Lys216 are also modified). Ebselen reacts with the sole Cys (Cys208) and ejects Zn2 from the active site. For each inhibitor, native UVPD-MS enabled simultaneous detection of the closing of a substrate-binding beta-hairpin loop, identification of covalently-modified residue(s), reporting of the metalation state of the enzyme, and in the case of ebselen, observation of the induction of partial disorder in the C-terminus of the protein. Owing to the ability of native UVPD-MS to track structural changes and metalation state with high sensitivity, we further used this method to evaluate the impact of mutations found in NDM clinical variants. Changes introduced by NDM-4 (M154L) and NDM-6 (A233V) are revealed to propagate through separate networks of interactions to direct zinc ligands, and the combination of these two mutations in NDM-15 (M154L, A233V) results in additive as well as additional structural changes. Insight from UVPD-MS helps to elucidate how distant mutations impact zinc affinity in the evolution of this antibiotic resistance determinant. UVPD-MS is a powerful tool capable of simultaneous reporting of ligand binding, conformational changes and metalation state of NDM, revealing structural aspects of ligand recognition and clinical variants that have proven difficult to probe.

Embolism resistance of different aged stems of a California oak species (Quercus douglasii): optical and microCT methods differ from the benchtop-dehydration standard.

Vulnerability of xylem to embolism is an important trait related to drought resistance of plants. Methods continue to be developed and debated for measuring embolism. We tested three methods (benchtop dehydration/hydraulic, micro-computed tomography (microCT) and optical) for assessing the vulnerability to embolism of a native California oak species (Quercus douglasii Hook. & Arn.), including an analysis of three different stem ages. All three methods were found to significantly differ in their estimates, with a greater resistance to embolism as follows: microCT > optical > hydraulic. Careful testing was conducted for the hydraulic method to evaluate multiple known potential artifacts, and none was found. One-year-old stems were more resistant than older stems using microCT and optical methods, but not hydraulic methods. Divergence between the microCT and optical methods from the standard hydraulic method was consistent with predictions based on known errors when estimating theoretical losses in hydraulic function in both microCT and optical methods. When the goal of a study is to describe or predict losses in hydraulic conductivity, neither the microCT nor optical methods are reliable for accurately constructing vulnerability curves of stems; nevertheless, these methods may be useful if the goal of a study is to identify embolism events irrespective of hydraulic conductivity or hydraulic function.

High-resolution computed tomography reveals dynamics of desiccation and rehydration in fern petioles of a desiccation-tolerant fern.

Desiccation-tolerant (DT) plants can dry past -100 MPa and subsequently recover function upon rehydration. Vascular DT plants face the unique challenges of desiccating and rehydrating complex tissues without causing structural damage. However, these dynamics have not been studied in intact DT plants. We used high resolution micro-computed tomography (microCT), light microscopy, and fluorescence microscopy to characterize the dynamics of tissue desiccation and rehydration in petioles (stipes) of intact DT ferns. During desiccation, xylem conduits in stipes embolized before cellular dehydration of living tissues within the vascular cylinder. During resurrection, the chlorenchyma and phloem within the stipe vascular cylinder rehydrated before xylem refilling. We identified unique stipe traits that may facilitate desiccation and resurrection of the vascular system, including xylem conduits containing pectin (which may confer flexibility and wettability); chloroplasts within the vascular cylinder; and an endodermal layer impregnated with hydrophobic substances that impede apoplastic leakage while facilitating the upward flow of water within the vascular cylinder. Resurrection ferns are a novel system for studying extreme dehydration recovery and embolism repair in the petioles of intact plants. The unique anatomical traits identified here may contribute to the spatial and temporal dynamics of water movement observed during desiccation and resurrection.

A Lysine-Targeted Affinity Label for Serine-ß-Lactamase Also Covalently Modifies New Delhi Metallo-ß-lactamase-1 (NDM-1).

The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-ß-lactamases hamper the development of wide-spectrum ß-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-ß-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-ß-lactamases have an analogous active-site Lys residue used to bind ß-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-ß-lactamases can also serve as a classical affinity label for New Delhi metallo-ß-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 µM) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-ß-lactamases.

Direct comparison of four methods to construct xylem vulnerability curves: Differences among techniques are linked to vessel network characteristics.

During periods of dehydration, water transport through xylem conduits can become blocked by embolism formation. Xylem embolism compromises water supply to leaves and may lead to losses in productivity or plant death. Vulnerability curves (VCs) characterize plant losses in conductivity as xylem pressures decrease. VCs are widely used to characterize and predict plant water use at different levels of water availability. Several methodologies for constructing VCs exist and sometimes produce different results for the same plant material. We directly compared four VC construction methods on stems of black cottonwood (Populus trichocarpa), a model tree species: dehydration, centrifuge, X-ray-computed microtomography (microCT), and optical. MicroCT VC was the most resistant, dehydration and centrifuge VCs were intermediate, and optical VC was the most vulnerable. Differences among VCs were not associated with how cavitation was induced but were related to how losses in conductivity were evaluated: measured hydraulically (dehydration and centrifuge) versus evaluated from visual information (microCT and optical). Understanding how and why methods differ in estimating vulnerability to xylem embolism is important for advancing knowledge in plant ecophysiology, interpreting literature data, and using accurate VCs in water flux models for predicting plant responses to drought.

Wood structure and function change with maturity: Age of the vascular cambium is associated with xylem changes in current-year growth.

Xylem vessel structure changes as trees grow and mature. Age- and development-related changes in xylem structure are likely related to changes in hydraulic function. We examined whether hydraulic function, including hydraulic conductivity and vulnerability to water-stress-induced xylem embolism, changed over the course of cambial development in the stems of 17 tree species. We compared current-year growth of young (1-4 years), intermediate (2-7 years), and older (3-10 years) stems occurring in series along branches. Diffuse and ring porous species were examined, but nearly all species produced only diffuse porous xylem in the distal branches that were examined irrespective of their mature xylem porosity type. Vessel diameter and length increased with cambial age. Xylem became both more conductive and more cavitation resistant with cambial age. Ring porous species had longer and wider vessels and xylem that had higher conductivity and was more vulnerable to cavitation; however, these differences between porosity types were not present in young stem samples. Understanding plant hydraulic function and architecture requires the sampling of multiple-aged tissues because plants may vary considerably in their xylem structural and functional traits throughout the plant body, even over relatively short distances and closely aged tissues.

Specificity of extended O-aryloxycarbonyl hydroxamates as inhibitors of a class C ß-lactamase.

Class C ß-lactamases have previously been shown to be efficiently inactivated by O-aryloxycarbonyl hydroxamates. O-Phenoxycarbonyl-N-benzyloxycarbonylhydroxylamine (1) and O-phenoxycarbonyl-N-(R)-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine (2), for example, were found to be effective inactivators. The present paper describes a structure-activity study of these molecules to better define the important structural elements for high inhibitory activity. The results show that a well-positioned hydrophobic element (which may interact with the Tyr221 residue of the enzyme) and a negatively charged element, e.g. a carboxylate group (which may interact with Arg204), are required for high reactivity with the enzyme. The new compounds were found to inactivate by forming a carbonyl cross-linked enzyme (probably Ser64OCONHLys 315) as for 1 rather than the inert hydroxamoyl derivative observed with 2.