Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production

The roles of two cytosolic maize glutamine synthetase isoenzymes (GS1), products of the Gln1-3 and Gln1-4 genes, were investigated by examining the impact of knockout mutations on kernel yield. In the gln1-3 and gln1-4 single mutants and the gln1-3 gln1-4 double mutant, GS mRNA expression was impaired, resulting in reduced GS1 protein and activity. The gln1-4 phenotype displayed reduced kernel size and gln1-3 reduced kernel number, with both phenotypes displayed in gln1-3 gln1-4. However, at maturity, shoot biomass production was not modified in either the single mutants or double mutants, suggesting a specific impact on grain production in both mutants. Asn increased in the leaves of the mutants during grain filling, indicating that it probably accumulates to circumvent ammonium buildup resulting from lower GS1 activity. Phloem sap analysis revealed that unlike Gln, Asn is not efficiently transported to developing kernels, apparently causing reduced kernel production. When Gln1-3 was overexpressed constitutively in leaves, kernel number increased by 30%, providing further evidence that GS1-3 plays a major role in kernel yield. Cytoimmunochemistry and in situ hybridization revealed that GS1-3 is present in mesophyll cells, whereas GS1-4 is specifically localized in the bundle sheath cells. The two GS1 isoenzymes play nonredundant roles with respect to their tissue-specific localization.     

Stenocarpella maydis (Berk.) Sutton Colonization of Maize Ears

A study was undertaken to determine the ramification of maize shank, cob and kernel tissues by Stenocarpella maydis. Trials consisting of inoculated and uninoculated treatments were planted at two localities. Shank, cob and kernels of each treatment were divided into segments and S. maydis colonization was determined. Infection of the pedicel portion of maize kernels was significantly higher than the apical portion. Preferential colonization of the embryo's of kernels was observed. Colonization of cobs occurred primarily at the attachment end of the cob, with sclerenchymatous tissues showing the greatest re-isolation frequency. Shank segments did not show significant differences in S. maydis re-isolation frequency, although a tendency for higher re-isolations was observed at the stalk-attachment end. It is concluded that S. maydis colonization occurs at the base of the ear with mycelial ramification toward the tip of the ear. The sclerenchyma and placentae were the primary colonized cob tissues. as were the embryos in the kernels.     

NUTRIENT CONTROLS ON NITROGEN UPTAKE AND METABOLISM BY NATURAL POPULATIONS AND CULTURES OF TRICHODESMIUM (CYANOBACTERIA)

The effects of inorganic nutrient (ammonium [NH₄ ⁺ ] and nitrate [NO₃ ⁻ ]) and amino acid (glutamate [glu] and glutamine [gln]) additions on rates of N₂ fixation, N uptake, glutamine synthetase (GS) activity, and concentrations of intracellular pools of gln and glu were examined in natural and cultured populations of Trichodesmium. Additions of 1 μM glu, gln, NO₃ ⁻ , or NH₄ ⁺ did not affect short-term rates of N₂ fixation. This may be an important factor that allows for continued N₂ fixation in oligotrophic areas where recycling processes are active. N₂ fixation rates decreased when nutrients were supplied at higher concentrations (e.g. 10 μM). Uptake of combined N (NH₄ ⁺ , NO₃ ⁻ , and amino acids) by Trichodesmium was stimulated by increased concentrations. For NO₃ ⁻ , proportional increases in NO₃ ⁻ uptake and decreases in N₂ fixation were observed when additions were made to cultures before the onset of the light period. GS activity did not change much in response to the addition of NH₄ ⁺ , NO₃ ⁻ , glu, or gln. GS is necessary for N metabolism, and the bulk of this enzyme pool may be conserved. Intracellular pools of glu and gln varied in response to 10 μM additions of NH₄ ⁺ , glu, or gln. Cells incubated with NH₄ ⁺ became depleted in intracellular glu and enriched with intracellular gln. The increase in the gln/glu ratio corresponded to a decrease in the rate of N₂ fixation. Although the gln/glu ratio decreased in cells exposed to the amino acids, there was only a corresponding decrease in N₂ fixation after the gln addition. The results presented here suggest that combined N concentrations on the order of 1 μM do not affect rates of N₂ fixation and metabolism, although higher concentrations (e.g. 10 μM) can. Moreover, these effects are exerted through products of NH₄ ⁺ assimilation rather than exogenous N, as has been suggested for other species. These results may help explain how cultures of Trichodesmium are able to simultaneously fix N₂ and take up NH₄ ⁺ and how natural populations continue to fix N₂ once combined N concentrations increase within a bloom.     

Amino acid metabolism in maize earshoots. Implications for assimilate preconditioning and nitrogen signaling

Nitrogen (N) is an essential requirement for kernel growth in maize (Zea mays); however, little is known about how N assimilates are metabolized in young earshoots during seed development. The objective of this study was to assess amino acid metabolism in cob and spikelet tissues during the critical 2 weeks following silking. Two maize hybrids were grown in the field for 2 years at two levels of supplemental N fertilizer (0 and 168 kg N/ha). The effects of the reproductive sink on cob N metabolism were examined by comparing pollinated to unpollinated earshoots. Earshoots were sampled at 2, 8, 14, and 18 d after silking; dissected into cob, spikelet, and/or pedicel and kernel fractions; then analyzed for amino acid profiles and key enzyme activities associated with amino acid metabolism. Major amino acids in the cob were glutamine (Gln), aspartic acid (Asp), asparagine (Asn), glutamate, and alanine. Gln concentrations dropped dramatically from 2 to 14 d after silking in both pollinated and unpollinated cobs, whereas all other measured amino acids accumulated over time in unpollinated spikelets and cobs, especially Asn. N supply had a variable effect on individual amino acid levels in young cobs and spikelets, with Asn being the most notably enhanced. We found that the cob performs significant enzymatic interconversions among Gln, alanine, Asp, and Asn during early reproductive development, which may precondition the N assimilate supply for sustained kernel growth. The measured amino acid profiles and enzymatic activities suggest that the Asn to Gln ratio in cobs may be part of a signal transduction pathway involving aspartate aminotransferase, Gln synthetase, and Asn synthetase to indicate plant N status for kernel development.     

The NIN‐like protein 5 (ZmNLP5) transcription factor is involved in modulating the nitrogen response in maize

Maize exhibits marked growth and yield response to supplemental nitrogen (N). Here, we report the functional characterization of a maize NIN‐like protein ZmNLP5 as a central hub in a molecular network associated with N metabolism. Predominantly expressed and accumulated in roots and vascular tissues, ZmNLP5 was shown to rapidly respond to nitrate treatment. Under limited N supply, compared with that of wild‐type (WT) seedlings, the zmnlp5 mutant seedlings accumulated less nitrate and nitrite in the root tissues and ammonium in the shoot tissues. The zmnlp5 mutant plants accumulated less nitrogen than the WT plants in the ear leaves and seed kernels. Furthermore, the mutants carrying the transgenic ZmNLP5 cDNA fragment significantly increased the nitrate content in the root tissues compared with that of the zmnlp5 mutants. In the zmnlp5 mutant plants, loss of the ZmNLP5 function led to changes in expression for a significant number of genes involved in N signalling and metabolism. We further show that ZmNLP5 directly regulates the expression of nitrite reductase 1.1 (ZmNIR1.1) by binding to the nitrate‐responsive cis‐element at the 5′ UTR of the gene. Interestingly, a natural loss‐of‐function allele of ZmNLP5 in Mo17 conferred less N accumulation in the ear leaves and seed kernels resembling that of the zmnlp5 mutant plants. Our findings show that ZmNLP5 is involved in mediating the plant response to N in maize.     

Cumulation of mycotoxins in maize cobs infected with<Emphasis Type="Italic">Fusarium gramihearum</Emphasis>

Maize cobs withFusarium ear rot were collected at 1986 season and five infected byFusarium graminearum were analyzed for presence of triohothecenes and zearalenone. Collected material was subsampled forFusarium damaged kernels and corresponding axial stems and healthy looking kernels. All investigated cobs contained deoxynivalenol (DON) (range 18.0–131.5 mg/kg) and zearalenone (ZEA) (range 0.38–2.17 mg/kg), in four cobs 15-acetyl-deoxynivalenol (15-AcDON) (range 5.2–6.2 mg/kg) was present and two cobs besides three all metabolites contained 3-acetyl-deoxynivalenol (3-AcD0N) (range 0.5–0.8 mg/kg).The average of individual toxins amount in axial stems: in mg/kg was equal to: DON — 110.36, ZEA — 4.57, 15-AcD0N — 16.66, and 3-AcD0N — 1.32.Fusarium damaged kernels contained in average the following amount (mg/kg) of: DON 77.00, ZEA 0.98, 15-AcD0N 3.78 and 3-AcD0N 0.06. Healthy looking kernels contained DON 1.96 mg/kg and ZEA 0.07 mg/kg only. Cooccurrence of 3-AcDON and 15-AcDON in two samples was an interesting finding. The amount of DON in total cob was highly correlated (r = 0.94) with percentage ofFusarium damaged kernels in given ear.     

Resistance of pepper germplasm to Meloidogyne incognita

In this work, we have evaluated the host suitability of 29 pepper genotypes of Capsicum annuum and 9 of the related cultivated species C. chinense (4), C. frutescens (4) and C. pubescens (1) to Meloidogyne incognita in field conditions. The presence/absence of resistance genes in the pepper germplasm were also assessed using PCR‐specific markers linked to the N, Me1‐Mech2, Me3‐Me4 and Me7‐Mech1 genes. Intraspecific variability for M. incognita resistance was found. According to gall index (GI) and reproduction index (RI) the most resistant genotypes, which may contribute to nematode management, include three of C. frutescens (Fru‐2, Fru‐3 and Fru‐4) and seven of C. annuum (Ca‐3, Ca‐4, Ca‐5, Ca‐11, Ca‐15, Ca‐24 and Ca‐25). No egg masses or eggs were found in Fru‐3 and Fru‐4 genotypes as occurred in the resistant controls ‘SCM’, ‘CH’ and ‘Charlot’. The amplification of markers linked to resistance genes in genotypes with a suitable degree of resistance, together with the differences found between genotypes with regard to the gene and/or number of amplified markers, make this germplasm a valuable tool for further characterisation and pepper breeding.     

Genetic and physiological analysis of germination efficiency in maize in relation to nitrogen metabolism reveals the importance of cytosolic glutamine synthetase

We have developed an approach combining physiology and quantitative genetics to enhance our understanding of nitrogen (N) metabolism during kernel germination. The physiological study highlighted the central role of glutamine (Gln) synthetase (GS) and Gln synthesis during this developmental process because a concomitant increase of both the enzyme activity and the amino acid content was observed. This result suggests that Gln is acting either as a sink for ammonium released during both storage protein degradation and amino acid deamination or as a source for amino acid de novo synthesis by transamination. In the two parental lines used for the quantitative genetics approach, we found that the increase in Gln occurred earlier in Io compared with F(2), a result consistent with its faster germinating capacity. The genetic study was carried out on 140 F6 recombinant inbred lines derived from the cross between F(2) and Io. Quantitative trait locus mapping identified three quantitative trait loci (QTLs) related to germination trait (T50, time at which 50% of the kernels germinated) that explain 18.2% of the phenotypic variance; three QTLs related to a trait linked to germination performance, kernel size/weight (thousand kernels weight), that explain 17% of the phenotypic variance; two QTLs related to GS activity at early stages of germination that explain 17.7% of the phenotypic variance; and one QTL related to GS activity at late stages of germination that explains 7.3% of the phenotypic variance. Coincidences of QTL for germination efficiency and its components with genes encoding cytosolic GS (GS1) and the corresponding enzyme activity were detected, confirming the important role of the enzyme during the germination process. A triple colocalization on chromosome 4 between gln3 (a structural gene encoding GS1) and a QTL for GS activity and T50 was found; whereas on chromosome 5, a QTL for GS activity and thousand kernels weight colocalized with gln4, another structural gene encoding GS1. This observation suggests that for each gene, the corresponding enzyme activity is of major importance for germination efficiency either through the size of the grain or through its faster germinating capacity. Consistent with the possible nonoverlapping function of the two GS1 genes, we found that in the parental line Io, the expression of Gln3 was transiently enhanced during the first hours of germination, whereas that of gln4 was constitutive.     

Genetic transformation of glutamine auxotrophy to prototrophy in the cyanobacterium Nostoc muscorum

Glutamine auxotrophic (Gln ^-) and l-methionine d,l-sulfoximine (MSX) resistant (MSX ^r) mutants of N. muscorum were isolated and characterized for nitrogen nutrition, nitrogenase activity, glutamine synthetase (GS) activity and glutamine amide, -keto-glutarate amido transferase (GOGAT) activity. The glutamine auxotroph was found to the GOGAT-containing GS-defective, incapable of growth with N₂ or NH ₄ ⁺ but capable of growth with glutamine as nitrogen source, thus, suggesting GS to be the primary enzyme of both ammonia assimilation and glutamine formation in the cyanobacterium. The results of transformation and reversion studies suggests that glutamine auxotrophy is the result of a mutation in the gln A gene and that gln A gene can be transferred from one strain to another by transformation.     

Indole-3-butyric acid synthesis in ecotypes and mutants of Arabidopsis thaliana under different growth conditions

Although IBA is a naturally occurring auxin, its role in plant development is still under debate. In this study a set of Arabidopsis mutants was used to analyze the biosynthesis of IBA in vitro. The mutants chosen for this study can be classified as: (1) involvement in auxin metabolism, transport or synthesis (amt1, aux1, ilr1, nit1, rib1, sur1, trp1-100); (2) other hormones possibly involved in the regulation of IBA synthesis (aba1, aba3, eto2, fae1, hls1, jar1); (3) photomorphogenesis (det1, det2, det3); and (4) root architecture (cob1, cob2, scr1). In addition, two transgenic lines overexpressing the IAA glucose synthase (iaglu) gene from maize were analyzed. The ecotypes No-0 and Wassilewskija showed the highest IBA synthetase activity under control conditions, followed by Columbia, Enkheim and Landsberg erecta. In the mutant lines IBA synthetase activity differed in most cases from the wild type, however no particular pattern of up- or down-regulation, which could be correlated to their possible function, was found. For rib1 mutant seedlings it was tested whether reduced IBA synthetase activity correlates with the endogenous IBA levels. Free IBA differed only depending on the culture conditions, but gave no clear correlation with IBA synthetase activity compared to the wild type. Since drought and osmotic stress as well as abscisic acid (ABA) application enhanced IBA synthesis in maize, it was tested whether IBA synthetase from Arabidopsis is also inducible by drought stress conditions. This was confirmed for the two ecotypes Col and Ler which showed different IBA synthetase activity when cultivated with various degrees of drought stress. IBA synthetase was also determined in photomorphogenic mutants under different light regimes. Induction of IBA synthetase in det1 and det3 plants was found under short day plus a red light pulse or in the dark, respectively. The results are discussed with respect to the functions of the mutated genes.     

Periphyton responds differentially to nutrients recycled in dissolved or faecal pellet form by the snail grazer Theodoxus fluviatilis

1. We aimed to separate the effects of grazers on periphyton via grazing from that of nutrient recycling from their faecal pellets. 2. We set up three different experimental treatments (snails/no snails/faecal pellets) and sampled over 16 days. The ‘snail’ treatment contained a low density (snail biomass c. 14 g^?2) of the gastropod grazer Theodoxus fluviatilis and the ‘faecal pellet’ treatment received the same amount of faecal pellets as were produced in the ‘snail’ treatment. Whereas the ‘faecal pellet’ treatment provided extra nutrients to periphyton from the faeces, the ‘snail’ treatment provided nutrients in the form of both faeces and in excreta. There was also direct grazing on periphyton in the ‘snail’ treatment. The ‘no snail’ was not grazed and received no nutrients in faeces or excreta. 3. We measured periphyton C, N and P content, chlorophyll‐a (chl‐a), primary production, bacterial biomass, bacterial production and bacterial respiratory activity. In the water column we measured dissolved inorganic N and soluble reactive P. 4. Snails increased the amount of dissolved inorganic N in the water. On day 16, the periphyton N : P ratio in the ‘faecal pellet’ treatment was lower, and periphyton P content was higher, than in the other two treatments. N : P ratios decreased over time in the ‘faecal pellet’ treatment. Primary and bacterial production were positively correlated in all treatments. 5. Algal chl‐a and primary production of periphyton per unit area and periphyton chl‐a : C ratios increased over the 16 day in the ‘snail’ treatment, and thus excretion of dissolved N by snails had a stronger positive effect on the periphyton community than N and P in faecal pellets. 6. Our data show that excretion and egestion can have different effects on periphyton, probably because of the higher proportion of dissolved N in excreta and the higher proportion of P recycled in faecal pellets. The relative effect of nutrients recycled in egesta or in excretions, probably depends on the form of nutrient limitation of the periphyton. Further, the different components of the periphyton matrix could react differently to the different forms of nutrient recycling. 7. We conclude that direct grazing effects are less important than nutrient effects when nutrients are limiting and grazing pressure is low. Further, the spatial separation of different grazing effects can lead to differences in periphyton production and nutrient stoichiometry. This might be an explanation for the patchiness of periphyton in nature.     

Fitness penalty in susceptible host is associated with virulence of Soybean mosaic virus on Rsv1‐genotype soybean: a consequence of perturbation of HC‐Pro and not P3

The multigenic Rsv1 locus in the soybean plant introduction (PI) ‘PI96983’ confers extreme resistance against the majority of Soybean mosaic virus (SMV) strains, including SMV‐N, but not SMV‐G7 and SMV‐G7d. In contrast, in susceptible soybean cultivars lacking a functional Rsv1 locus, such as ‘Williams82’ (rsv1), SMV‐N induces severe disease symptoms and accumulates to a high level, whereas both SMV‐G7 and SMV‐G7d induce mild symptoms and accumulate to a significantly lower level. Gain of virulence by SMV‐N on Rsv1‐genotype soybean requires concurrent mutations in both the helper‐component proteinase (HC‐Pro) and P3 cistrons. This is because of the presence of at least two resistance (R) genes, probably belonging to the nucleotide‐binding leucine‐rich repeat (NB‐LRR) class, within the Rsv1 locus, independently mediating the recognition of HC‐Pro or P3. In this study, we show that the majority of experimentally evolved mutational pathways that disrupt the avirulence functions of SMV‐N on Rsv1‐genotype soybean also result in mild symptoms and reduced accumulation, relative to parental SMV‐N, in Williams82 (rsv1). Furthermore, the evaluation of SMV‐N‐derived HC‐Pro and P3 chimeras, containing homologous sequences from virulent SMV‐G7 or SMV‐G7d strains, as well as SMV‐N‐derived variants containing HC‐Pro or P3 point mutation(s) associated with gain of virulence, reveals a direct correlation between the perturbation of HC‐Pro and a fitness penalty in Williams82 (rsv1). Collectively, these data demonstrate that gain of virulence by SMV on Rsv1‐genotype soybean results in fitness loss in a previously susceptible soybean genotype, this being a consequence of mutations in HC‐Pro, but not in P3.     

“Ecological Armageddon” – more evidence for the drastic decline in insect numbers

The efficient use of nitrogen by crops can minimise environmental risks and maximise returns to farmers. Under organic farming systems, this can be achieved by adjusting the fertilisation management and/or using genetic variability. Seven durum wheat (Triticum durum) cultivars and three emmer (Triticum dicoccum) cultivars were assessed under an organic farming system over a non‐consecutive 4‐year period (2005–11) in Foggia (southern Italy). The objectives were to investigate the agronomic and qualitative characteristics, and to evaluate the agronomic efficiency and adaptability according to three N fertilisation levels (0, 40, 80 kg N ha^?1). A split‐plot design was used, with three replications in each year. Nine traits were investigated: heading time, plant height, seed yield, number of spikes m^?2, harvest index, specific weight, 1000‐seed weight, and protein and gluten contents. Increasing N to 80 kg ha^?1 increased plant height (+7.3%), seed yield (+22.1%), spike density (+16.6%), and protein (+9.8%) and gluten (+2.1%) contents. The emmer genotypes and the oldest durum wheat ‘Cappelli’ showed the highest protein and gluten contents (mean, 13.9%, 11.2%, respectively). The maximum N agronomic efficiency [AE(N)] and N recovery efficiency [RE(N)] were seen for the modern durum wheat cultivars under 40 kg ha^?1 N treatment: ‘Duilio’, ‘Iride’ and ‘Varano’. The significant correlation between AE(N) and RE(N) and the year of release of the durum wheat cultivars (r = 0.53, P < 0.05, F = 4.7 for AE(N) and r = 0.57, P < 0.01, F = 5.7 for RE(N), respectively; n = 14) showed that the genetic breeding of this species for high grain yield is associated with an increase in the efficiency of N fertiliser use under organic farming. The highly significant effect for the Genotype × Environment interaction (F = 18.1, P < 0.001) of seed yield indicate the possibility to select for stable genotypes across environments. Based on the additive main effects and multiplicative interaction analysis, and the yield stability index, the varieties ‘Iride’ and ‘Varano’ show high‐stability responses and good seed yields under all N fertiliser treatments. Therefore, these varieties can be recommended for organic farming systems in Mediterranean areas.     

DcMYB113, a root‐specific R2R3‐MYB,conditions anthocyanin biosynthesis and modification in carrot

Purple carrots, the original domesticated carrots, accumulate highly glycosylated and acylated anthocyanins in root and/or petiole. Previously, a quantitative trait locus (QTL) for root‐specific anthocyanin pigmentation was genetically mapped to chromosome 3 of carrot. In this study, an R2R3‐MYB gene, namely DcMYB113, was identified within this QTL region. DcMYB113 expressed in the root of ‘Purple haze’, a carrot cultivar with purple root and nonpurple petiole, but not in the roots of two carrot cultivars with a purple root and petiole (Deep purple and Cosmic purple) and orange carrot ‘Kurodagosun’, which appeared to be caused by variation in the promoter region. The function of DcMYB113 from ‘Purple haze’ was verified by transformation in ‘Cosmic purple’ and ‘Kurodagosun’, resulting in anthocyanin biosynthesis. Transgenic ‘Kurodagosun’ carrying DcMYB113 driven by the CaMV 35S promoter had a purple root and petiole, while transgenic ‘Kurodagosun’ expressing DcMYB113 driven by its own promoter had a purple root and nonpurple petiole, suggesting that root‐specific expression of DcMYB113 was determined by its promoter. DcMYB113 could activate the expression of DcbHLH3 and structural genes related to anthocyanin biosynthesis. DcUCGXT1 and DcSAT1, which were confirmed to be responsible for anthocyanins glycosylation and acylation, respectively, were also activated by DcMYB113. The WGCNA identified several genes co‐expressed with anthocyanin biosynthesis and the results indicated that DcMYB113 may regulate anthocyanin transport. Our findings provide insight into the molecular mechanism underlying root‐specific anthocyanin biosynthesis and further modification in carrot and even other root crops.     

Enhancement of cadmium accumulation in sweet sorghum as affected by nitrate

• The Cadmium (Cd)‐polluted soils are is an increasing concern worldwide. Phytoextraction of Cd pollutants by high biomass plants, such as sweet sorghum, is considered an environmentally‐friendly, cost‐effective and sustainable strategy for remediating this problem. Nitrogen (N) is a macronutrient essential for plant growth, development and stress resistance. Nevertheless, how nitrate, as an important form of N, affects Cd uptake, translocation and accumulation in sweet sorghum is still unclear.
• In the present study, a series of nitrate levels (N1, 0.5 mm ; N2, 2 mm ; N3, 4 mm ; N4, 8 mm and N5, 16 mm ) with or without added 5 μm CdCl₂ treatment in sweet sorghum was investigated hydroponically.
• The results indicate that Cd accumulation in the aboveground parts of sweet sorghum was enhanced by optimum nitrate supply, resulting from both increased dry weight and Cd concentration. Although root‐to‐shoot Cd translocation was not enhanced by increased nitrate, some Cd was transferred from cell walls to vacuoles in leaves. Intriguingly, expression levels of Cd uptake and transport genes, SbNramp1, SbNramp5 and SbHMA3, were not closely related to increased Cd as affected by nitrate supply. The expression of SbNRT1.1B in relation to nitrate transport showed an inverted ‘U’ shape with increasing nitrate levels under Cd stress, which was in agreement with trends in Cd concentration changes in aboveground tissues.
• Based on the aforementioned results, nitrate might regulate Cd uptake and accumulation through expression of SbNRT1.1B rather than SbNramp1, SbNramp5 or SbHMA3, the well‐documented genes related to Cd uptake and transport in sweet sorghum.