Significance of root hairs for plant performance under contrasting field conditions and water deficit.

BACKGROUND AND AIMS: Previous laboratory studies have suggested selection for root hair traits in future crop breeding to improve resource use efficiency and stress tolerance. However, data on the interplay between root hairs and open-field systems, under contrasting soils and climate conditions, are limited. As such, this study aims to experimentally elucidate some of the impacts that root hairs have on plant performance on a field scale. METHODS: A field experiment was set up in Scotland for two consecutive years, under contrasting climate conditions and different soil textures (i.e. clay loam vs. sandy loam). Five barley (Hordeum vulgare) genotypes exhibiting variation in root hair length and density were used in the study. Root hair length, density and rhizosheath weight were measured at several growth stages, as well as shoot biomass, plant water status, shoot phosphorus (P) accumulation and grain yield. KEY RESULTS: Measurements of root hair density, length and its correlation with rhizosheath weight highlighted trait robustness in the field under variable environmental conditions, although significant variations were found between soil textures as the growing season progressed. Root hairs did not confer a notable advantage to barley under optimal conditions, but under soil water deficit root hairs enhanced plant water status and stress tolerance resulting in a less negative leaf water potential and lower leaf abscisic acid concentration, while promoting shoot P accumulation. Furthermore, the presence of root hairs did not decrease yield under optimal conditions, while root hairs enhanced yield stability under drought. CONCLUSIONS: Selecting for beneficial root hair traits can enhance yield stability without diminishing yield potential, overcoming the breeder's dilemma of trying to simultaneously enhance both productivity and resilience. Therefore, the maintenance or enhancement of root hairs can represent a key trait for breeding the next generation of crops for improved drought tolerance in relation to climate change.

Root growth in field-grown winter wheat: Some effects of soil conditions, season and genotype.

This work compared root length distributions of different winter wheat genotypes with soil physical measurements, in attempting to explain the relationship between root length density and soil depth. Field experiments were set up to compare the growth of various wheat lines, including near isogenic lines (Rht-B1a Tall NIL and Rht-B1c Dwarf NIL) and wheat lines grown commercially (cv. Battalion, Hystar Hybrid, Istabraq, and Robigus). Experiments occurred in two successive years under rain fed conditions. Soil water content, temperature and penetrometer resistance profiles were measured, and soil cores taken to estimate vertical profiles of pore distribution, and root number with the core-break method and by root washing. Root length distributions differed substantially between years. Wetter soil in 2014/2015 was associated with shallower roots. Although there was no genotypic effect in 2014/2015, in 2013/2014 the dwarf wheat had the most roots at depth. In the shallower layers, some wheat lines, especially Battalion, seemed better at penetrating non-structured soil. The increase in penetrometer resistance with depth was a putative explanation for the rapid decrease in root length density with depth. Differences between the two years in root profiles were greater than those due to genotype, suggesting that comparisons of different genotypic effects need to take account of different soil conditions and seasonal differences. We also demonstrate that high yields are not necessarily linked to resource acquisition, which did not seem to be limiting in the low yielding dwarf NIL.

Sap fluxes from different parts of the rootzone modulate xylem ABA concentration during partial rootzone drying and re-wetting.

Previous studies with partial rootzone drying (PRD) irrigation demonstrated that alternating the wet and dry parts of the rootzone (PRD-Alternated) increased leaf xylem ABA concentration ([X-ABA]leaf) compared with maintaining the same wet and dry parts of the rootzone (PRD-Fixed). To determine the relative contributions of different parts of the rootzone to this ABA signal, [X-ABA]leaf of potted, split-root tomato (Solanum lycopersicum) plants was modelled by quantifying the proportional water uptake from different soil compartments, and [X-ABA]leaf responses to the entire pot soil-water content (θpot). Continuously measuring soil-moisture depletion by, or sap fluxes from, different parts of the root system revealed that water uptake rapidly declined (within hours) after withholding water from part of the rootzone, but was rapidly restored (within minutes) upon re-watering. Two hours after re-watering part of the rootzone, [X-ABA]leaf was equally well predicted according to θpot alone and by accounting for the proportional water uptake from different parts of the rootzone. Six hours after re-watering part of the rootzone, water uptake by roots in drying soil was minimal and, instead, occurred mainly from the newly irrigated part of the rootzone, thus [X-ABA]leaf was best predicted by accounting for the proportional water uptake from different parts of the rootzone. Contrary to previous results, alternating the wet and dry parts of the rootzone did not enhance [X-ABA]leaf compared with PRD-Fixed irrigation. Further work is required to establish whether altered root-to-shoot ABA signalling contributes to the improved yields of crops grown with alternate, rather than fixed, PRD.

Methods to estimate changes in soil water for phenotyping root activity in the field.

BACKGROUND AND AIMS: There is an urgent need to develop new high throughput approaches to phenotype roots in the field. Excavating roots to make direct measurements is labour intensive. An alternative to excavation is to measure soil drying profiles and to infer root activity. METHODS: We grew 23 lines of wheat in 2013, 2014 and 2015. In each year we estimated soil water profiles with electrical resistance tomography (ERT), electromagnetic inductance (EMI), penetrometer measurements and measurements of soil water content. We determined the relationships between the measured variable and soil water content and matric potential. RESULTS: We found that ERT and penetrometer measurements were closely related to soil matric potential and produced the best discrimination between wheat lines. We found genotypic differences in depth of water uptake in soil water profiles and in the extent of surface drying. CONCLUSIONS: Penetrometer measurements can provide a reliable approach to comparing soil drying profiles by different wheat lines, and genotypic rankings are repeatable across years. EMI, which is more sensitive to soil water content than matric potential, and is less effective in drier soils than the penetrometer or ERT, nevertheless can be used to rapidly screen large populations for differences in root activity.

Genetic analysis of rootstock-mediated nitrogen (N) uptake and root-to-shoot signalling at contrasting N availabilities in tomato.

Selecting rootstocks for high nitrogen acquisition ability may allow decreased N fertilizer application without reducing tomato yields, minimizing environmental nitrate pollution. A commercial hybrid tomato variety was grafted on a genotyped population of 130 recombinant inbred lines (RILs) derived from Solanum pimpinellifolium, and compared with self- and non-grafted controls under contrasting nitrate availabilities (13.8 vs 1.0mM) in the nutrient solution. Grafting itself altered xylem sap composition under N-sufficient conditions, particularly Na+ (8.75-fold increase) concentration. N deprivation decreased shoot dry weight by 72.7% across the grafted RIL population, and one RIL rootstock allowed higher total leaf N content than the best of controls, suggesting more effective N uptake. Sixty-two significant QTLs were detected by multiple QTL mapping procedure for leaf N concentration (LNC), vegetative growth, and the xylem sap concentrations of Mn and four phytohormone groups (cytokinins, gibberellins, salicylic acid and jasmonic acid). Only three LNC QTLs could be common between nitrogen treatments. Clustering of rootstock QTLs controlling LNC, leaf dry weight and xylem sap salicylic acid concentration in chromosome 9 suggests a genetic relationship between this rootstock phytohormone and N uptake efficiency. Some functional candidate genes found within 2 Mbp intervals of LNC and hormone QTLs are discussed.

Elution of interferon beta from blue sepharose by poly(vinylpyrrolidone).

Interferon beta produced by a cell line (MRC-5) of human embryonic lung fibroblasts adsorbs to Blue Sepharose CL-6B and may be dissociated at neutral pH using a buffer containing poly(vinylpyrrolidone) and sodium chloride. Poly(vinylpyrrolidone) of Mr 2500 or of Mr 25 000 is equally efficient on a w/w basis. The effect of substituting volatile ammonium salts for the sodium chloride has been investigated. The benefits of the use of poly(vinylpyrrolidone) compared with the use of the commonly-used dissociating agent, 1,2-ethanediol, are discussed. It is concluded that interferon preparations eluted by poly(vinylpyrrolidone) from Blue Sepharose may be suitable for clinical use with minimal additional processing or formulation.

Systematic method for the detection of potential lambda Cro-like DNA-binding regions in proteins.

We have developed and tested a systematic method for the location and statistical evaluation of potential DNA-binding regions of the lambda Cro type in protein sequences. Using this approach to examine proteins expected to contain such regions, we have been able to compile a statistically homogeneous master set of 37 lambda Cro-like DNA-binding domains. Examination of a protein database revealed other prokaryotic proteins that are similar to this lambda Cro-like group. There are also many DNA-binding proteins that are not found to be significantly similar to the lambda Cro group, consistent with previous suggestions that different types of protein sequence may be able to achieve a similar mode of binding and that there exist other modes of sequence-specific DNA-binding. A useful feature of the method is that it can be applied without a computer.

Control of gene expression in the temperate coliphage 186. VIII. Control of lysis and lysogeny by a transcriptional switch involving face-to-face promoters.

The lysogenic and early lytic operons of the temperate coliphage 186 are transcribed divergently. Primer extension mapping of the 5' ends of these in vivo transcripts showed that the rightward lytic promoter, pR, and the leftward lysogenic promoter, pL, are arranged face-to-face, with their transcripts overlapping by 60 bases. We examined the control of transcription from pR and pL using galK as a reporter gene. The product of the lysogenic cI gene strongly repressed pR transcription while allowing pL transcription. The product of the lytic apl gene (formerly CP75) strongly repressed pL transcription while allowing pR transcription. Thus, the cI-pR-pL-apl region functioned as a transcriptional switch, determining whether transcription was lytic or lysogenic. Also, the cI gene product was able to stimulate pL, possibly by alleviating an inhibition of pL transcription caused by convergent transcription from pR. Other consequences of the face-to-face promoter arrangement are discussed.

Control of gene expression in the P2-related template coliphages. III. DNA sequence of the major control region of phage 186.

The PstI fragment (65.5% to 77.4%) of coliphage 186, known genetically to encode the major control genes, has been sequenced, and an analysis performed to assess coding capacity, transcription-translation signals, and to identify any other significant features. Our analysis indicates that the region encodes: seven genes, including the int and cI genes, which overlap, the late control gene B, and two genes, named CP75 and CP76, encoding potential DNA-binding proteins; a promoter pB and terminator tB for the rightward transcription of the B gene, and we predict the existence of this transcript in a lysogen; a promoter pL and terminator tL for leftward transcription that encodes the int and cI genes, and represents the presumed lysogenic transcript; a promoter pR for rightward transcription to give the presumed (early) lytic transcript that is overlapping and convergent with the lysogenic transcript; and finally, a potential operator site for repressor binding in the region of the pR promoter. Preliminary evidence is presented to support this analysis.

NMR analysis of main-chain conformational preferences in an unfolded fibronectin-binding protein.

A 130-residue fragment of the Staphylococcus aureus fibronectin-binding protein has been found to exist in a highly unfolded conformation at neutral pH. Measurement of experimental NMR 3JHNalpha coupling constants provides evidence for individual residues having distinct main-chain conformational preferences that are dependent both on the amino acid concerned and on neighbouring residues in the sequence. Analysis shows that these variations in the populations of individual residues can be explained in detail in terms of statistical distributions of conformational states derived from the protein data base. In particular, when the preceding residue has a beta-branched or aromatic side-chain, a significant increase occurs in the population of the less sterically restricted b region of phi,psi space. The results indicate that the local structure of the fibronectin binding protein in solution, under conditions where it displays full activity, approximates very closely to a statistical random coil structure. This may be an important feature in the biological role of this and other polypeptides involved in protein-protein interactions.

Strategies for targeting complement inhibitors in ischaemia/reperfusion injury.

A transplanted organ suffers inherently from an ischaemic insult and subsequent reperfusion injury. The severity of such early events is thought to influence the success of the transplant procedure, not only in the immediate post-transplant period, but also to predispose the graft to both acute and chronic rejection. In this paper, we review the influence of the complement system upon ischaemia,reperfusion injury. The recognition of the involvement of complement has led to novel strategies to try to modulate ischaemia/reperfusion injury, some of which we have summarized. Finally, we note our own strategy to target complement inhibition in ischaemic tissues.

Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells.

We have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libraries of human genomic DNA. Several related genomic clones were isolated. One of these, a cosmid clone, carried approx. 40 kb of human DNA. Mapping experiments indicate that the region containing the protein-coding exons is approx. 20 kb in length. The cosmid, containing the t-PA gene and the aminoglycosyl-3'-phosphotransferase dominant-selection marker, was introduced into mouse L cells. Approximately half of the transformants were shown to produce human t-PA. We demonstrated that the fibrinolytic t-PA activity could be specifically quenched by anti-t-PA antibody and that the recombinant t-PA was of similar size (by SDS-polyacrylamide gel electrophoresis) to the t-PA produced by the human Bowes melanoma cell line. Our results suggest that the cosmid clone carries the whole t-PA coding region together with the regulatory elements necessary for its expression.

Large scale, rapid purification of recombinant tissue-type plasminogen activator.

Recombinant tissue-type plasminogen activator (rt-PA) from cultures of a genetically manipulated Bowes melanoma cell line (TRBM6) was purified in batches of average volume 451 using an autoclavable, reusable, continuous chromatography system comprising zinc chelate-Sepharose CL4B and lysine-Sepharose CL4B. After eight successive purifications the rt-PA was ultrafiltered to yield a preparation containing 4.9 mg protein/ml and 2.7 X 10(6) IU/ml. Analysis by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue R250 showed major protein bands at Mr = 63,000 and 65,000; most of the material was in the 1-chain form. The potential usefulness of a simple, rapid continuous chromatography system that can be operated under aseptic conditions is discussed.

Isolation and preliminary characterisation of active B-chain of recombinant tissue-type plasminogen activator.

Purified 2-chain recombinant tissue-type plasminogen activator (t-PA) was reduced under mild conditions - 10 mM dithiothreitol/5 degrees C/1.5 h - and the two chains were separated by chromatography on lysine Sepharose. The t-PA B chain was fully active as determined by its activity towards the chromogenic substrate S-2288 (H-D-ile-pro-arg p-nitroanilide). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing or non-reducing conditions revealed a single polypeptide at Mr = 35,000 or 29,000 respectively. In addition, under non-reducing conditions a fibrinolytic band at apparent Mr = 29,000 was present after fibrin zymography. The N-terminal sequence was confirmed as ile-lys-gly. The t-PA B chain had a specific amidolytic activity, using S-2288, of 170,000 to 210,000 SU/mg protein. (This compares to a specific activity of the native 2-chain t-PA of 170,000 SU/mg). It resembles urokinase-type plasminogen activator in its inability to be stimulated by fibrin and its dose response on human fibrin plates. However, t-PA B-chain was stimulated to almost the same extent as t-PA by poly-D-lysine. The isoelectric points, at pH 5.6 and 5.7, fall outside the range generally quoted for t-PA preparations (pH 7.8-8.8).

Isolation, identification and pharmacokinetic properties of human tissue-type plasminogen activator species: possible localisation of a clearance recognition site.

Purified preparations of recombinant tissue-type plasminogen activator (t-PA) from the recombinant Bowes melanoma cell line TRBM6 were shown to contain multiple species of plasminogen activator. Using a combination of chromatography on Sephadex G25, Sephadex G75 and Heparin Sepharose CL6B we have isolated two fibrinolytically active species, which, under non-reduced SDS PAGE, have apparent Mr = 38,000 and 56,000. Double immunodiffusion studies indicated that both species were closely related to both the t-PA B chain and t-PA itself. N-terminal sequencing identified the Mr = 38,000 species as ala160- t-PA (essentially delta FGKI t-PA) and the Mr = 56,000 species as ser1-tyr2-gln3-glyx-cys51 t-PA (delta F t-PA), the latter probably produced by alternative splicing of the t-PA gene. The pharmacokinetic properties of N,N dimethyl-4-aminobenzoyl (DAB) derivatives of these activators and native t-PA were determined in the guinea pig. Whereas DAB----delta F t-PA showed a similar, rapid plasma disappearance profile to that of DAB----t-PA, DAB----delta FGKI t-PA was cleared significantly slower. These results suggest that a rapid clearance recognition site resides on either the growth factor or kringle 1, or both, domains of t-PA.

Interaction of a plasmin A-chain/t-PA B-chain hybrid enzyme with plasma inhibitors in vivo and in vitro.

A hybrid plasminogen activator consisting of the "A" chain of plasmin linked to the "B" chain of rt-PA was inhibited in vitro in human and guinea pig plasmas 4 to 5-fold more rapidly than its parent activator, two-chain t-PA. Using zymographic and autoradiographic techniques together with the use of immunodepleted plasma the major inhibitor was identified as alpha-2-antiplasmin. The pharmacokinetic profile of the hybrid in guinea pigs was determined by two different methods: disappearance of fibrinolytic activity and removal of radiolabelled hybrid from the circulation. Fibrinolytic activity was cleared rapidly via inhibitory mechanisms, whilst radiolabelled material was cleared considerably more slowly due to the formation of hybrid-inhibitor complexes. When the active site of the hybrid was reversibly acylated inhibitory mechanisms were evaded and a prolonged pharmacokinetic profile of activity was observed.

Slow clearance of acylated, hybrid thrombolytic enzymes.

Two hybrid plasminogen activators, plasmin A-chain/t-PA B-chain and plasmin A-chain/u-PA B-chain have been synthesized and purified in sufficient yield to permit measurement of clearance in small laboratory animals. Each hybrid enzyme was reversibly acylated at the active centre to allow the pharmacokinetic profile to be followed using an activity-based method without interference from plasma inhibitors. The acylated plasmin/u-PA hybrid had a clearance half-life (t1/2) in guinea pigs of approximately 80 min, whereas acyl u-PA had a t1/2 of 3 min. The pharmacokinetic profile of the acylated plasmin/t-PA hybrid was measured in guinea pigs, rats and rabbits; the half-lives in all three species were 60-80 min compared to half-lives of acylated, native t-PA that were in the range 0.5-1.0 min. Thus, plasmin A-chain-containing, acylated hybrid enzymes are cleared some 30- to 100-fold more slowly than the acylated parent activators.

Increased yield of human tissue-type plasminogen activator obtained by means of recombinant DNA technology.

Extra copies of the human tissue-type plasminogen activator (t-PA) gene were introduced into the Bowes melanoma cell line. We obtained a recombinant cell line (TRBM6) which secretes approximately ten-fold more t-PA than the parent cell line. The identity of the plasminogen activator made by the new cell line was confirmed by sizing on sodium dodecyl sulphate polyacrylamide gels and by specific quenching using anti-t-PA antibody. We estimate that the recombinant line produces t-PA at a rate of approximately 3 pg/cell/24 hr and that t-PA accumulates in the harvest medium at a rate of approximately 4000 International t-PA Units/ml/24 hr.

The use of active centre acylation to control the pharmacokinetic profile of a recombinant chimaeric plasminogen activator.

Recombinant hybrid plasminogen activators consisting of the "A" chain of plasminogen linked to the "B" chain of t-PA that are inhibited rapidly by plasma protease inhibitors have recently been described (Robinson et al. Circulation 1992; 86: 548-552). We have now shown that following bolus administration of native hybrid to guinea pigs, fibrinolytic activity was cleared rapidly from the circulation. Active centre acylation appeared to protect the hybrid from inhibition and allowed material to circulate as potentially active species for prolonged periods. Clearance rates of a range of acyl derivatives of the hybrid were 7-35-fold slower than for native hybrid and 20-100-fold slower than for t-PA. Clearance rates were influenced markedly by deacylation rate, such that clearance half-life correlated well with deacylation half-life. We have thus shown that it is feasible to control the pharmacokinetic profile of a recombinant hybrid plasminogen activator over a wide range by selection of an appropriate acyl group for attachment to the active site. Such control is not possible with plasminogen activators that are cleared predominantly by mechanisms other than inhibition.