ALBERT

Notes: Abstract. Although nitrate reductase (NR, EC 1.6.6.1) is thought to control the rate of nitrate assimilation, mutants with 40–45% of wildtype (WT) NR activity (NRA) grow as fast as the WT. We have investigated how tobacco (Nicotiana tabacum L. cv. Gatersleben) mutants with one or two instead of four functional nia genes compensate. (i) The nia transcript was higher in the leaves of the mutants. However, the diurnal rhythm was retained in the mutants, with a maximum at the end of the night and a strong decline during the photoperiod. (ii) Nitrate reductase protein and NRA rose to a maximum after 3–4 h light in WT leaves, and then decreased by 50–60% during the second part of the photoperiod and the first part of the night. Leaves of mutants contained 40–60% less NR protein and NRA after 3–4 h illumination, but NR did not decrease during the photoperiod. At the end of the photoperiod the WT and the mutants contained similar levels of NR protein and NRA. (iii) Darkening led to a rapid inactivation of NR in the WT and the mutants. However, in the mutants, this inactivation was reversed after 1–3 h darkness. Calyculin A prevented this reversal. When magnesium was included in the assay to distinguish between the active and inactive forms of NR, mutants contained 50% more activity than the WT during the night. Conversion of [15N]-nitrate to organic compounds in leaves in the first 6 h of the night was 60% faster in the mutants than in the WT. (iv) Growth of WT plants in enhanced carbon dioxide prevented the decline of NRA during the second part of the photoperiod, and led to reactivation of NR in the dark. (v) Increased stability of NR in the light and reversal of dark-inactivation correlated with decreased levels of glutamine in the leaves. When glutamine was supplied to detached leaves it accelerated the breakdown of NR, and led to inactivation of NR, even in the light. (vi) Diurnal changes were also investigated in roots. In the WT, the amount of nia transcript rose to a maximum after 4 h illumination and then gradually decreased. The amplitude of the changes in transcript amount was smaller in roots than in leaves, and there were no diurnal changes in NRA. In mutants, nia transcript levels were high through the photoperiod and the first part of the night. The NRA was 50% lower during the day but rose during the night to an activity almost as high as in the WT. The rate of [15N]-nitrate assimilation in the roots of the mutants resembled that in the WT during the first 6 h of the night. (vii) Diurnal changes were also compared in Nia30(145) transformants with very low NRA, and in nitrate-deficient WT plants. Both sets of plants had similar low growth rates. Nitrate reductase did not show a diurnal rhythm in leaves or roots of Nia30(145), the leaves contained very low glutamine, and NR did not inactivate in the dark. Nitrate-deficient WT plants were watered each day with 0.2 mM nitrate. After watering, there was a small peak of nia transcript, NR protein and NRA and, slightly later, a transient increase of glutamine and other amino acids in the leaves. During the night glutamine was low, and NR did not inactivate. In the roots, there was a very marked increase of nitrate, nia transcript and NRA 2–3 h after the daily watering with 0.2 mM nitrate. (viii) It is concluded that WT plants have excess capacity for nitrate assimilation. They only utilise this potential capacity for a short time each day, and then down-regulate nitrate assimilation in response, depending on the conditions, to accumulation of the products of nitrate assimilation or exhaustion of external nitrate. Genotypes with a lower capacity for nitrate assimilation compensate by increasing expression of NR and weakening the feedback regulation, to allow assimilation to continue for a longer period each day.

Notes: Abstract. Tobacco (Nicotiana tabacum L.) plants were used to study connections between deficiency in boron and nitrate reduction. Boron deficiency caused a substantial decrease in shoot and, particularly, root weights that resulted in a notably high shoot/root ratio in comparison to boron-sufficient plants. One of the most important effects caused by boron deficiency was the strong decrease in leaf nitrate content. Leaf contents of magnesium, calcium and, especially, potassium also declined under this deficiency, but nitrate content decreased in a higher proportion than these cations. Nitrate reductase (EC 1.6.6.1) activity of boron-deficient plants declined from the beginning of the light period; this decline did not occur in boron-sufficient plants. This fact could be attributed to the faster decrease in transcript levels of Nia, the nitrate reductase structural gene, during the light period in boron-deficient plants. Leaf protein content of boron-deficient plants also declined in the course of light periods. Boron deficiency caused an appreciable accumulation of hexoses and sucrose in leaves. This build-up of soluble sugars might correct the osmotic imbalance elicited by the low content of nitrate and cations in plants subjected to boron deficiency. Boron-deficient plants had much higher starch contents than boron-sufficient ones, and there was an inverse relationship between the contents of nitrate and starch in leaves.

Notes: Abstract  The streak disease has a major effect on maize in sub-Saharan Africa. Various genetic factors for resistance to the virus have been identified and mapped in several populations; these factors derive from different sources of resistance. We have focused on the Réunion island source and have recently identified several factors in the D211 line. A second very resistant line, CIRAD390, was crossed to the same susceptible parent, B73. The linkage map comprised 124 RFLP markers, of which 79 were common with the D211×B73 map. A row-column design was used to evaluate the resistance to maize streak virus (MSV) of 191 F2:3 families under artificial infestation at two locations: Harare (Zimbabwe) and in Réunion island. Weekly ratings of resistance were taken and disease incidence and severity calculated. QTL analyses were conducted for each scoring date and for the integration over time of the disease scores, of incidence, and of severity. Heritability estimates (71–98%) were as high as for the D211×B73 population. Eight QTLs were detected on chromosomes 1, 2, 3, 5 (two QTLs), 6, 8, and 10. The chr1-QTL explained the highest proportion of phenotypic variation, about 45%. The QTLs on chromosomes 1, 2, and 10 were located in the same chromosomal bin as QTLs for MSV resistance in the D211×B73 population. In a simultaneous fit, QTLs explained together 43–67% of the phenotypic variation. The QTLs on chromosomes 3, 5, and 6 appeared to be specific for one or the other component of the resistance. For the chr3-QTL, resistance was contributed by the susceptible parent. There were significant QTL × environment interactions for some of the variables studied, but QTLs were stable in the two environments. They also appeared to be stable over time. Global gene action ranged from partial dominance to overdominance, except for disease severity. Some additional putative QTLs were also detected. The major QTL on chromosome 1 seemed to be common to the other sources of resistance, namely Tzi4, a tolerant line from IITA, and CML202 from CIMMYT. However, the distribution of the other QTLs within the genome revealed differences in Réunion germplasm and across these other resistance sources. This diversity is of great importance when considering the durability of the resistance.

Notes: Abstract Drought is an important climatic phenomenon which, after soil infertility, ranks as the second most severe limitation to maize production in developing countries. When drought stress occurs just before or during the flowering period, a delay in silking is observed, resulting in an increase in the length of the anthesis-silking interval (ASI) and in a decrease in grain yield. Selection for reduced ASI in tropical open-pollinated varieties has been shown to be correlated with improved yields under drought stress. Since efficient selection for drought tolerance requires carefully managed experimental conditions, molecular markers were used to identify the genomic segments responsible for the expression of ASI, with the final aim of developing marker-assisted selection (MAS) strategies. An F2population of 234 individuals was genotyped at 142 loci and F3 families were evaluated in the field under several water regimes for male flowering (MFLW), male sterility (STER), female flowering (FFLW) and ASI. The genetic variance of ASI increased as a function of the stress intensity, and the broad-sense heritabilites of MFLW, FFLW and ASI were high under stress conditions, being 86%, 82% and 78%, respectively. Putative quantitative trait loci (QTLs) involved in the expression of MFLW and/or FFLW under drought were detected on chromosomes 1, 2, 4, 5, 8, 9 and 10, accounting for around 48% of the phenotypic variance for both traits. For ASI, six putative QTLs were identified under drought on chromosomes 1, 2, 5, 6, 8 and 10, and together accounted for approximately 47% of the phenotypic variance. Under water stress conditions, four QTLs were common for the expression of MFLW and FFLW, one for the expression of ASI and MFLW, and four for the expression of ASI and FFLW. The number of common QTLs for two traits was related to the level of linear correlation between these two traits. Segregation for ASI was found to be transgressive with the drought-susceptible parent contributing alleles for reduced ASI (4 days) at two QTL positions. Alleles contributed by the resistant line at the other four QTLs were responsible for a 7-day reduction of ASI. These four QTLs represented around 9% of the linkage map, and were stable over years and stress levels. It is argued that MAS based on ASI QTLs should be a powerful tool for improving drought tolerance of tropical maize inbred lines.

Notes: Abstract  Maize streak virus (MSV) disease may cause significant grain yield reductions in maize in Africa. Réunion island maize germplasm is a proven source of strong resistance. Its genetic control was investigated using 123 RFLP markers in an F2 population of D211 (resistant) × B73 (susceptible). This population of 165 F2:3 families was carefully evaluated in Harare (Zimbabwe) and in Réunion. Artificial infestation was done with viruliferous leafhoppers. Each plant was rated weekly six times after infestation on a 1–9 scale previously adjusted by image analysis. QTL analyses were conducted for each scoring date, and for the areas under the disease, incidence and severity progress curves. The composite interval mapping method used allowed the estimation of the additive and dominance effects and QTL × environment interactions. Heritabilities ranged from 73% to 98%, increasing with time after infestation. Resistance to streak virus in D211 was provided by one region on chromosome 1, with a major effect, and four other regions on chromosomes 2, 3 (two regions) and 10, with moderate or minor effects. Overall, they explained 48–62% of the phenotypic variation for the different variables. On chromosome 3, one of the two regions seemed to be more involved in early resistance, whereas the second was detected at the latest scoring date. Other QTLs were found to be stable over time and across environments. Mild QTL × environment interactions were detected. Global gene action appeared to be partially dominant, in favor of resistance, except at the earliest scoring dates, where it was additive. From this population, 32 families were chosen, representing the whole range of susceptibility to MSV. They were tested in Réunion against three MSV clones, along with a co-inoculation of two of them. Virulence differences between clones were significant. There were genotype × clone interactions, and these were more marked for disease incidence than for severity. Although these interactions were not significant for the mean disease scores, it is suggested that breeders should select for completely resistant genotypes.

Notes: Abstract  In most maize-growing areas yield reductions due to drought have been observed. Drought at flowering time is, in some cases, the most damaging. In the experiment reported here, trials with F3 families, derived from a segregating F2 population, were conducted in the field under well-watered conditions (WW) and two other water-stress regimes affecting flowering (intermediate stress, IS, and severe stress, SS). Several yield components were measured on equal numbers of plants per family: grain yield (GY), ear number (ENO), kernel number (KNO), and 100-kernel weight (HKWT). Correlation analysis of these traits showed that they were not independent of each other. Drought resulted in a 60% decrease of GY under SS conditions. By comparing yield under WW and SS conditions, the families that performed best under WW conditions were found to be proportionately more affected by stress, and the yield reductions due to SS conditions were inversely proportional to the performance under drought. Moreover, no positive correlation was observed between a drought-tolerance index (DTI) and yield under WW conditions. The correlation between GY under WW and SS conditions was 0.31. Therefore, in this experiment, selection for yield improvement under WW conditions only, would not be very effective for yield improvement under drought. Quantitative trait loci (QTLs) were identified for GY, ENO and KNO using composite interval mapping (CIM). No major QTLs, expressing more then 13% of the phenotypic variance, were detected for any of these traits, and there were inconsistencies in their genomic positions across water regimes. The use of CIM allowed the evaluation of QTL-by-environment interactions (Q×E) and could thus identify “stable” QTLs CIMMYT, Apartado Postal 6-641, 06600 Mexico D.F., Mexico across drought environments. Two such QTLs for GY, on chromosomes 1 and 10, coincided with two stable QTLs for KNO. Moreover, four genomic regions were identified for the expression of both GY and the anthesis-silking interval (ASI). In three of these, the allelic contributions were for short ASI and GY increase, while for that on chromosome 10 the allelic contribution for short ASI corresponded to a yield reduction. From these results, we hypothesize that to improve yield under drought, marker-assisted selection (MAS) using only the QTLs involved in the expression of yield components appears not to be the best strategy, and neither does MAS using only QTLs involved in the expression of ASI. We would therefore favour a MAS strategy that takes into account a combination of the “best QTLs” for different traits. These QTLs should be stable across target environments, represent the largest percentage possible of the phenotypic variance, and, though not involved directly in the expression of yield, should be involved in the expression of traits significantly correlated with yield, such as ASI.

Notes: Summary We investigated the inducibility of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), and glutamine synthetase (GS; EC 6.3.1.2) isoforms in cotyledons of 7-day-old seedlings of sunflower (Helianthus annuus L.) in relation to light, nitrogen source (NO 3 − , NO 2 − or NH 4 + ), and the involvement of plastids. Nitrate was absolutely (and specifically) required for NR induction, and stimulated more effectively than NO 2 − or NH 4 + the synthesis of NiR and chloroplastic GS (GS2) over the constitutive levels present in N-free-grown seedlings. In vivo inhibition of NR activity by tungsten application to seedlings and measurements of tissue NO 3 − concentration indicate that NO 3 − -dependent enzyme induction is elicited by NO 3 − per se and not by a product of its assimilatory reduction, e.g., NO 2 − or NH 4 + . In the presence of NO 3 − , light remarkably enhanced the appearance of NR, NiR, and GS2, while the activity of the cytosolic GS isoform (GS1) was adversely affected. Cycloheximide suppressed much more efficiently than chloramphenicol the light- and NO 3 − -dependent increase of GS2 activity, indicating that sunflower chloroplastic GS is synthesized on cytoplasmic 80S ribosomes. When the plastids were damaged by photooxidation in cotyledons made carotenoid-free by application of norflurazon, the positive action of light and NO 3 − on the appearance of NR, NiR, and GS2 isoform was greatly abolished. Therefore, it is suggested that intact chloroplasts are required for the inductive effect of light and NO 3 − and/or for the accumulation of newly formed enzymes in the organelle.

Notes: Abstract Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3 − null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.

Notes: The electron self-exchange rate constant for the Type 1 blue copper protein umecyanin from horseradish roots has been determined as 6.1 × 103 M-1 S-1 at pH 7.5, I = 0.100 M, 25°C by an NMR line-broadening method. The value obtained is one of the lower self-exchange rate constants determined for this class of protein; this is attributed to the presence of positively charged residues near to the electron-transfer site. The self-exchange rate constants calculated by means of a Marcus analysis of data for the cross-reactions (25°C) of umecyanin with azurin and cytochrome c551 (both from Pseudomonas aeruginosa) are substantially less at 8.0M-1 S-1 and 13.9M-1S-1, respectively, and are independent of pH in the range 7.0-8.0, I = 0.100M. The discrepancy between the self-exchange rate constants obtained by these two different methods can be rationalised if it is assumed that umecyanin reacts with the two proteins employed in the cross-reaction studies through the same site, but that this site is different from that used for the self-exchange process. A comparison of the primary structure of umecyanin with those of other Type 1 copper proteins has revealed that a glutamine rather than a methionine is likely as the fourth ligand of Cu at the active site. Other comparisons are made with stellacyanin, and the electron-transfer reactivity of the two proteins is discussed.

Notes: A convenient diastereoselective synthesis of an immediate precursor for a rapid access to a variety of homo-carbocyclic nucleosides, is described. The synthetic scheme involves a high yielding new type of Pd(0) catalyzed cyclization of a suitable hydrazine derivative together with a stereospecific OsO4 triggered dihydroxylation step.