Integrating genetic information into plant breeding programmes: how will we produce varieties from molecular variation,using bioinformatics?

The development of new varieties of crop plants is ongoing for plant breeders and progress since the Green Revolution has been steady, if not dramatic. With the recent sequencing of Arabidopsis thaliana and of rice the development of both physical and informational resources has entered a new phase. This paper examines the state of plant bioinformatics as it is now and as it is likely to develop in the future. It also looks rather further forward to what crop scientists might want from bioinformatics, before examining the likely physiological targets for sustainability traits and the prospects for their improvement in wheat. Wheat is taken as the focus crop because it is potentially one of the most dif. Cult to work with in molecular terms, both because of its large hexaploid genome size and because of its considerable genetic distance from the most information rich plant species, Arabidopsis. Finally, we examine how these tools might be used to track down the underlying genes controlling sustainability traits and how these may then be exploited in plant breeding programmes using marker‐ assisted selection.     

Instability of familial spongiform encephalopathy-related prion mutants

We examined the influence of D177N (D178N in humans) mutation on the conformational stability of the S2 region of moPrP^C with varying pHs by using the SDSL-ESR technique. The ESR spectrum of D177N at pH 7.5 was narrower than that of Y161R1, referred to as WT^∗. The ESR spectrum of D177N did not change when pH in the solution decreased to pH 4.0. Our results suggested that the disappearance of a salt bridge (D177-R163) induced the increase in the instability of S2 region. Moreover, the line shape of the ESR spectrum obtained from H176S neighboring the salt bridge linked to the S2 region was similar to D177N. These results indicate that the protonation of H176 is strongly associated with the stability of S2 region. These findings are important for understanding the mechanism by which the disruption of the salt bridge in the S2 region forms the pathogenic PrP^Sc structure in hereditary prion disease.     

Crystal structure and mutational study of a unique SpoU family archaeal methylase that forms 2'-O-methylcytidine at position 56 of tRNA

The conserved cytidine residue at position 56 of tRNA contributes to the maintenance of the L-shaped tertiary structure. aTrm56 catalyzes the 2′-O-methylation of the cytidine residue in archaeal tRNA, using S-adenosyl-L-methionine. Based on the amino acid sequence, aTrm56 is the most distant member of the SpoU family. Here, we determined the crystal structure of Pyrococcus horikoshii aTrm56 complexed with S-adenosyl-L-methionine at 2.48 Å resolution. aTrm56 consists of the SPOUT domain, which contains the characteristic deep trefoil knot, and a unique C-terminal β-hairpin. aTrm56 forms a dimer. The S-adenosyl-L-methionine binding and dimerization of aTrm56 were similar to those of the other SpoU members. A structure-based sequence alignment revealed that aTrm56 conserves only motif II, among the four signature motifs. However, an essential Arg16 residue is located at a novel position within motif I. Biochemical assays showed that aTrm56 prefers the L-shaped tRNA to the λ form as its substrate.     

Compensatory Evolution of a WW Domain Variant Lacking the Strictly Conserved Trp Residue

Replacement of conserved amino acid residues during evolution of proteins can lead to divergence and the formation of new families with novel functions, but is often deleterious to both protein structure and function. Using the WW domain, we experimentally examined whether and to what degree second-site mutations can compensate for the reduction of function and loss of structure that accompany substitution of a strictly conserved amino acid residue. The W17F mutant of the WW domain, with substitution of the most strictly conserved Trp residue, is known to lack a specific three-dimensional structure and shows reduced binding affinity in comparison to the wild type. To obtain second-site revertants, we performed a selection experiment based on the proline-rich peptide (PY ligand) binding affinity using the W17F mutant as the initial sequence. After selection by ribosome display, we were able to select revertants that exhibited a maximum ninefold higher affinity to the PY ligand than the W17F mutant and showed an even better affinity than the wild type. In addition, we found that the functional restoration resulted in increased binding specificity in selected revertants, and the structures were more compact, with increased amounts of secondary structure, in comparison to the W17F mutant. Our results suggest that the defective structure and function of the proteins caused by mutations in highly conserved residues occurring through divergent evolution not only can be restored but can be further improved by compensatory mutations.     

ATP synthesis without R210 of subunit a in the Escherichia coli ATP synthase

Interactions between subunit a and oligomeric subunit c are essential for the coupling of proton translocation to rotary motion in the ATP synthase. A pair of previously described mutants, R210Q/Q252R and P204T/R210Q/Q252R [L.P. Hatch, G.B. Cox and S.M. Howitt, The essential arginine residue at position 210 in the a subunit of the Escherichia coli ATP synthase can be transferred to position 252 with partial retention of activity, J. Biol. Chem. 270 (1995) 29407-29412] has been constructed and further analyzed. These mutants, in which the essential arginine of subunit a, R210, was switched with a conserved glutamine residue, Q252, are shown here to be capable of both ATP synthesis by oxidative phosphorylation, and ATP-driven proton translocation. In addition, lysine can replace the arginine at position 252 with partial retention of both activities. The pH dependence of ATP-driven proton translocation was determined after purification of mutant enzymes, and reconstitution into liposomes. Proton translocation by the lysine mutant, and to a lesser extent the arginine mutant, dropped off sharply above pH 7.5, consistent with the requirement for a positive charge during function. Finally, the rates of ATP synthesis and of ATP-driven proton translocation were completely inhibited by treatment with DCCD (N,N′-dicyclohexylcarbodiimide), while rates of ATP hydrolysis by the mutants were not significantly affected, indicating that DCCD modification disrupts the F₁-F_o interface. The results suggest that minimal requirements for proton translocation by the ATP synthase include a positive charge in subunit a and a weak interface between subunit a and oligomeric subunit c.     

Microflora in the crop of adult Dusky-billed Parrotlets (Forpus modestus)

We isolated aerobic and anaerobic facultative bacteria inhabiting the crop of adult Dusky-billed Parrotlets (Forpus modestus). We looked for bacteria capable of hydrolyzing starch, the most abundant polysaccharide in seeds. We compared our results with bacteria isolated from the crop of three species of doves with granivorous–frugivorous diet and three carnivore birds. Forpus modestus has 10⁷–10⁸ of colony formation units (CFU); these values were higher by one to three orders of magnitude compared with those observed in the other species studied. Bacillus pumilus, one of the most abundant bacteria isolated in F. modestus (6.03 × 10⁶ CFU), was capable of hydrolyzing starch. We found higher diversity and abundance of bacteria in granivorous than in carnivorous birds or birds without a developed crop. Additionally, we found yeasts in the three species of doves. These findings suggest microbial activity in the crop, although its importance in food digestion needs to be determined.     

Effects of foliar application of BAP on source and sink strength in four six-rowed barley (Hordeum vulgare L.) cultivars

A field experiment was conducted to investigate the effects of foliar application of a synthetic cytokinin (BAP) on source and sink strength of four different six-rowed barley (Hordeum vulgare L.) cultivars. Different spraying treatments consisting of spraying on whole plant, spraying only on leaves and spraying only on ears started at anthesis and continued for 7 days. One additional spraying was carried out on late period of grain filling. Results showed that spraying only on leaves did not affect ear weight, grain yield and 1,000-grain weight, while the two other treatments increased all above mentioned traits. Neither of treatments affected stem weight, biological yield and contribution of stem reserves in grain filling. Exogenous cytokinin did not increase photosynthetic rate and chlorophyll content in treated leaves until late period of grain filling, although there was no significant increase in final grain weight due to late application of BAP. Our results suggested that effects of foliar application of BAP were mostly due to increased sink size soon after anthesis and increased sink demand probably met by current photosynthesis of organs other than leaves, like ear green tissues. An erratum to this article is available at .     

组织工程的临床研究——组织工程连载之五

组织工程研究涉及的临床科室包括骨科、普外科、五官科、康复医学科、泌尿科、口腔颌面外科、神经外科、整形外科、胸外科、眼科、肝胆外科、血管外科;涉及的组织器官有:神经组织、肝脏组织、角膜组织、膀胱组织、血液、韧带、耳朵、生殖道、手、脂肪、乳房、心脏、肾脏、胰腺、管状组织(用于建造肠管、食管、气管、血管、肾和尿道等)等.其中皮肤组织、软骨组织、骨组织等的研究应用较为成熟.从最初工程化组织或器官的立项研究到最终批准临床应用,这是一个漫长的过程,需要众多不同学科的科研人员共同努力.随着基础研究和临床应用的深入发展,现代组织工程正在成为治疗组织、器官衰竭的有效疗法和辅助手段.     

Conventionally Tilled and Permanent Raised Beds with Different Crop Residue Management: Effects on Soil C and N Dynamics

Conservation tillage in its version of permanent bed planting under zero-tillage with crop residue retention has been proposed as an alternative wheat production system for northwest Mexico. However, little is known about the dynamics of C and N in soils under wheat/maize on permanent beds (PB) where straw was burned, removed, partly removed or retained, as opposed to conventionally tilled beds (CTB) where straw was incorporated. We investigated the dynamics of soil C and N and normalized difference vegetative index (NDVI) crop values in zero-tilled PB and CTB after 26 successive maize and wheat crops. Organic C and total N were respectively, 1.15 and 1.17 times greater in PB with straw partly removed and with straw retained on the surface, than in CTB with straw incorporated. Organic C and total N were 1.10 times greater in soils with 300 kg N ha⁻¹ added than in unfertilized soil. Cumulative production of CO₂ was lower under CTB with straw incorporated than under PB treatments, and CO₂ production increased with increments in inorganic fertilizer. The N-mineralization rate was 1.18 times greater than in unamended soils when 150 kg inorganic N ha⁻¹ was applied, and 1.48 times greater when 300 kg inorganic N ha⁻¹ was added. The N-mineralization rate was significantly (1.66 times) greater in PB where the straw was burned or retained on the surface than in CTB where the straw was incorporated, but significantly (1.25 times) lower than in PB with straw partly removed. The NDVI values reached a maximum 56 days after planting and decreased thereafter. The NDVI for unfertilized soil were similar for CTB with straw incorporated, PB with straw partly removed, and PB with straw retained on the surface, but significantly lower for PB with straw burned and PB with straw removed. In soils to which 150 or 300 kg N ha⁻¹ was added, NDVI was significantly lower for PB with straw burned than for other treatments. Among other things, this suggests the utility of rotating maize or wheat with crops whose residues have lower C–N ratios, thus avoiding immobilization of large amounts of N for extended periods. PB with residue burning, however, is an unsustainable practice leading to low crop performance and soil and environmental degradation.     

Phosphorus benefits from grain-legume crops to subsequent maize grown on acid soils of southern Cameroon

We conducted field experiments over 2 years on two acid soils of southern Cameroon to test whether efficient uptake and use of phosphorus (P) from less available sources by grain legume genotypes could benefit subsequent rotational maize. We grew two crops each year. For the first crop we grew 4 genotypes of soybean and of cowpea, plus maize. For the second crop we grew maize. The first crops were fertilized with 0, 90 kg P ha⁻¹ as phosphate rock (PR) or 30 kg P ha⁻¹ as triple super phosphate (TSP). P application highly significantly increased shoot dry matter, P uptake, N₂ fixation and grain yields of the grain legumes with TSP generally more effective than PR. Two of the soybean and two of the cowpea genotypes were more efficient at using P. Only the P-efficient soybean and cowpea genotypes increased subsequent maize yields. Yields of the subsequent maize grown in rotation were significantly correlated with shoot P uptake for which the quantity of P applied with the crop residues of the pre-crop appeared to be a major factor. We also grew the grain legumes in nutrient solutions and measured organic acid-anion exudation from roots, root-surface phosphatase-activity, and root morphological characteristics. Enhanced exudation of organic acid anions from roots of P-deprived plants might have contributed to the P acquisition efficiency under field conditions of the P-efficient cowpea genotypes and one of the P-efficient soybean genotypes. A higher activity of root-surface acid phosphatase might have been important for the other P-efficient soybean genotype. The results show, that the potential positive rotational effect of cowpea and soybean on the acid, highly P-sorbing soils of southern Cameroon depends on breeding and using P-efficient genotypes when sparingly soluble and suboptimal rates of soluble P fertilizers are used. Section Editor: N. J. Barrow