Differential Expression of Proteins in Response to Molybdenum Deficiency in Winter Wheat Leaves Under Low-Temperature Stress

Molybdenum (Mo) is an essential micronutrient for plants. To obtain a better understanding of the molecular mechanisms of cold resistance enhanced by molybdenum application in winter wheat, we applied a proteomic approach to investigate the differential expression of proteins in response to molybdenum deficiency in winter wheat leaves under low-temperature stress. Of 13 protein spots that were identified, five spots were involved in the light reaction of photosynthesis, five were involved in the dark reaction of photosynthesis, and three were highly involved in RNA binding and protein synthesis. Before the application of cold stress, four differentially expressed proteins between the Mo deficiency (?Mo) vs. Mo application (+Mo) comparison are involved in carbon metabolism and photosynthetic electron transport. After 48 h of cold stress, nine differentially expressed proteins between the ?Mo vs. +Mo comparison are involved in carbon metabolism, photosynthetic electron transport, RNA binding, and protein synthesis. Under ?Mo condition, cold stress induced a more than twofold decrease in the accumulation of six differential proteins including ribulose bisphosphate carboxylase large-chain precursor, phosphoglycerate kinase, cp31BHv, chlorophyll a/b-binding protein, ribulose bisphosphate carboxylase small subunit, and ribosomal protein P1, whereas under +Mo condition cold stress only decreased the expression of RuBisCO large subunit, suggesting that Mo application might contribute to the balance or stability of these proteins especially under low-temperature stress and that Mo deficiency has greater influence on differential protein expression in winter wheat after low-temperature stress. Further investigations showed that Mo deficiency decreased the concentrations of chlorophyll a, chlorophyll b, and carotenoids; the maximum net photosynthetic rate; the apparent quantum yield; and carboxylation efficiency, even before the application of the cold stress, although the decrease rates were greater after 48 h of cold treatment, which is consistent with changes in the expressions of differential proteins in winter wheat under low-temperature stress. These findings provide some new evidence that Mo might be involved in the light and dark reaction of photosynthesis and protein synthesis.     

The Arabidopsis Nitrate Transporter NRT1.7, Expressed in Phloem,Is Responsible for Source-to-Sink Remobilization of Nitrate

Several quantitative trait locus analyses have suggested that grain yield and nitrogen use efficiency are well correlated with nitrate storage capacity and efficient remobilization. This study of the Arabidopsis thaliana nitrate transporter NRT1.7 provides new insights into nitrate remobilization. Immunoblots, quantitative RT-PCR, β-glucuronidase reporter analysis, and immunolocalization indicated that NRT1.7 is expressed in the phloem of the leaf minor vein and that its expression levels increase coincidentally with the source strength of the leaf. In nrt1.7 mutants, more nitrate was present in the older leaves, less ¹⁵NO₃⁻ spotted on old leaves was remobilized into N-demanding tissues, and less nitrate was detected in the phloem exudates of old leaves. These data indicate that NRT1.7 is responsible for phloem loading of nitrate in the source leaf to allow nitrate transport out of older leaves and into younger leaves. Interestingly, nrt1.7 mutants showed growth retardation when external nitrogen was depleted. We conclude that (1) nitrate itself, in addition to organic forms of nitrogen, is remobilized, (2) nitrate remobilization is important to sustain vigorous growth during nitrogen deficiency, and (3) source-to-sink remobilization of nitrate is mediated by phloem.     

OsNRAMP3 Is a Vascular Bundles-Specific Manganese Transporter That Is Responsible for Manganese Distribution in Rice

Manganese (Mn) is an essential trace element for plants. Recently, the genes responsible for uptake of Mn in plants were identified in Arabidopsis and rice. However, the mechanism of Mn distribution in plants has not been clarified. In the present study we identified a natural resistance-associated macrophage protein (NRAMP) family gene in rice, OsNRAMP3, involved in Mn distribution. OsNRAMP3 encodes a plasma membrane-localized protein and was specifically expressed in vascular bundles, especially in phloem cells. Yeast complementation assay showed that OsNRAMP3 is a functional Mn-influx transporter. When OsNRAMP3 was absent, rice plants showed high sensitivity to Mn deficiency. Serious necrosis appeared on young leaves and root tips of the OsNRAMP3 knockout line cultivated under low Mn conditions, and high Mn supplies could rescue this phenotype. However, the necrotic young leaves of the knockout line possessed similar levels of Mn to the wild type, suggesting that the necrotic appearance was caused by disturbed distribution of Mn but not a general Mn shortage. Additionally, compared with wild type, leaf Mn content in osnramp3 plants was mostly in older leaves. We conclude that OsNRAMP3 is a vascular bundle-localized Mn-influx transporter involved in Mn distribution and contributes to remobilization of Mn from old to young leaves.     

YSL16 Is a Phloem-Localized Transporter of the Copper-Nicotianamine Complex That Is Responsible for Copper Distribution in Rice

Cu is an essential element for plant growth, but the molecular mechanisms of its distribution and redistribution within the plants are unknown. Here, we report that Yellow stripe-like16 (YSL16) is involved in Cu distribution and redistribution in rice (Oryza sativa). Rice YSL16 was expressed in the roots, leaves, and unelongated nodes at the vegetative growth stage and highly expressed in the upper nodes at the reproductive stage. YSL16 was expressed at the phloem of nodes and vascular tissues of leaves. Knockout of this gene resulted in a higher Cu concentration in the older leaves but a lower concentration in the younger leaves at the vegetative stage. At the reproductive stage, a higher Cu concentration was found in the flag leaf and husk, but less Cu was present in the brown rice, resulting in a significant reduction in fertility in the knockout line. Isotope labeling experiments with ⁶⁵Cu showed that the mutant lost the ability to transport Cu-nicotianamine from older to younger leaves and from the flag leaf to the panicle. Rice YSL16 transported the Cu-nicotianamine complex in yeast. Taken together, our results indicate that Os-YSL16 is a Cu-nicotianamine transporter that is required for delivering Cu to the developing young tissues and seeds through phloem transport.     

Interactive effects of molybdenum and phosphorus fertilizers on photosynthetic characteristics of seedlings and grain yield of Brassica napus

A pot experiment with acid yellow–brown soil was conducted to investigate the interactive effects of molybdenum (Mo) and phosphorus (P) fertilizers on the photosynthetic characteristics of seedlings and grain yield of Brassica napus which is sensitive to soil P and Mo deficiency. Both Mo and P fertilizers were applied at three levels (0 mg Mo kg^?1, 0.15 mg Mo kg^?1, 0.30 mg Mo kg^?1 soil; 0 mg P kg^?1, 80 mg P kg^?1, 160 mg P kg^?1 soil). The results showed that P fertilizer application increased grain yield, soluble sugar concentrations of seedling leaves, DM and P accumulation of seedling shoots of Brassica napus in the absence or presence of Mo fertilizer. In contrast, Mo fertilizer increased these parameters only in the presence of P fertilizer. Mo accumulation in shoots, chlorophyll concentrations and net photosynthesis rate (P _n) of seedling leaves were increased by both Mo and P fertilizers, particularly with the combination of the two fertilizers. The results also showed that the Mo and P fertilizers increased photosynthetic rate through two different mechanisms, with Mo increasing photosynthetic activity of mesophyll cells, and P increasing stomatal conductance. The results demonstrate that there was a synergetic effect on photosynthesis and grain yield between Mo and P fertilizers and it is conducive for Brassica napus growth to co-apply the two fertilizers.     

Reducing the potential copper toxicity of concentrates to sheep by the use of molybdenum and sulphur supplements

A high copper (Cu) diet (45.3 μg Cu/g DM) was given to three groups of animals, ♂ or ♀ Scottish Blackface and ♂ Finnish Landrace lambs, without added molybdenum (Mo), or with 2, 4, 8 or 16 mg Mo/kg DM added in a 3 × 5 factorial experiment lasting 18–27 weeks. Sodium sulphate, providing 2 g S/kg, was added with each Mo supplement.Six of the nine lambs not given supplementary Mo + S died of Cu poisoning but those given Mo + S survived. Histological evidence of subclinical hepato-toxicity was found in Mo + S supplemented lambs but it decreased in severity as the level of added Mo increased. Plasma aspartate amino-transferase (PAAT) concentrations were elevated in unsupplemented lambs from week 9 and in lambs given 2 mg Mo/kg from week 12 but they remained normal in lambs given 4–16 mg Mo/kg DM. Successive increments in dietary Mo reduced the increase in liver Cu after 18–20 weeks from 1450 to 735, 483, 445 and 131 μg/g DM. The proportion of ingested Cu (y%) retained in the liver was related to dietary Mo (x, mg/kg DM) by the equation y = 2.6 ? 1.66 log x ± 0.21 (r = 0.98; 2 d.f.).Finnish Landrace lambs retained 50% less Cu in their livers, had lower PAAT levels and showed less histological evidence of liver damage than ♂ Scottish Blackface lambs. The latter had higher PAAT levels and a higher mortality from Cu poisoning than ♀ Scottish Blackface lambs although the two sexes retained similar proportions of ingested Cu in their livers.The results are discussed in relation to the practical use of Mo + S to prevent Cu poisoning in sheep.     

Effect of Cu and Mo on Cu/Mo ratio and their concentration in different organs of cowpeas (Vigna sinensis L.)

In a Typic Torripsamments (loamy sand) soil, 4 levels each of Cu and Mo,viz, 0, 5, 10 and 20; and 0, 0.5, 1.0 and 2.0 ppm, respectively, were added to study Cu–Mo relationship in cowpeas in a screen-house. Application of Mo reduced Cu content andvice versa in leaves, stems and roots of cowpeas. Stems accumulated more of Mo and Cu than leaves and roots, showing thereby, that it might be the site of interaction for Mo and Cu. Molybdenum increased to a toxic level in plant (for animals) following its application. Cu/Mo ratio was narrowed with Mo and widened with Cu addition. It was either more than 21 or less which suggested that Mo would cause Cu deficiency and Cu in the absence of Mo would be toxic to ruminants.     

Phosphorus remobilization from rice flag leaves during grain filling: an RNA‐seq study

The physiology and molecular regulation of phosphorus (P) remobilization from vegetative tissues to grains during grain filling is poorly understood, despite the pivotal role it plays in the global P cycle. To test the hypothesis that a subset of genes involved in the P starvation response are involved in remobilization of P from flag leaves to developing grains, we conducted an RNA‐seq analysis of rice flag leaves during the preremobilization phase (6 DAA) and when the leaves were acting as a P source (15 DAA). Several genes that respond to phosphate starvation, including three purple acid phosphatases (OsPAP3, OsPAP9b and OsPAP10a), were significantly up‐regulated at 15 DAA, consistent with a role in remobilization of P from flag leaves during grain filling. A number of genes that have not been implicated in the phosphate starvation response, OsPAP26, SPX‐MFS1 (a putative P transporter) and SPX‐MFS2, also showed expression profiles consistent with involvement in P remobilization from senescing flag leaves. Metabolic pathway analysis using the KEGG system suggested plastid membrane lipid synthesis is a critical process during the P remobilization phase. In particular, the up‐regulation of OsPLDz2 and OsSQD2 at 15 DAA suggested phospholipids were being degraded and replaced by other lipids to enable continued cellular function while liberating P for export to developing grains. Three genes associated with RNA degradation that have not previously been implicated in the P starvation response also showed expression profiles consistent with a role in P mobilization from senescing flag leaves.     

Effect of Molybdenum and Inorganic Nitrogen on Molybdenum Redistribution in Black Gram (Vigna mungo L. Hepper) with Particular Reference to Seed Fill

Seeds used to plant a crop may contain sufficient molybdenum(Mo) to prevent subsequent Mo deficiency in the crop even whenthey are sown on Mo deficient soils. However, little is knownabout either the sources of the Mo acquired by the seed, orthe timing of its redistribution during seed development. Aglasshouse experiment was set up to examine the effect of Mosupply and nitrogen source on the redistribution of Mo withinblack gram, from full flowering to seed maturity. Treatmentscomprised two sources of N (symbiotic N₂fixation, NH₄NO₃), twolevels of Mo supply [nil (-Mo), 0.64 mg Mo kg^-1soil (+Mo)] andfour harvests (full flowering, early pod setting, late pod fillingand seed maturity). The redistribution of Mo in black gram wasexamined by determining changes over time in the content ofMo in plant parts at each growth stage. Molybdenum supply and the plant growth stage strongly affectedthe redistribution of Mo to the seed. In -Mo plants relianton symbiotic N₂fixation, Mo redistributed from roots, stemsand leaves was the only source of Mo for reproductive developmentsince, from full flowering until maturity, there was no netincrease in whole plant Mo. For pod and early seed development,the roots were the major source of Mo in -Mo plants. After latepod filling, nodules replaced roots as the major source of Mofor seed fill in -Mo plants. By contrast, for +Mo plants relianton symbiotic N₂fixation, Mo taken up from the soil after fullflowering could have supplied nearly 50% of the seed Mo. Themajor sources of Mo for seed filling in +Mo plants were middlestem leaves during early podding, and middle stems and pod wallsfrom late podding. Supplying NH₄NO₃to plants from sowing had little effect on Modistribution or redistribution in +Mo black gram plants. However,in -Mo plants it accelerated the loss of Mo from middle stemsand their leaves compared to nodulated plants. Black gram; Vigna mungo L. Hepper; distribution; molybdenum; nitrogen; nodules; redistribution; seed fill     

Efficient phloem remobilization of Zn protects apple trees during the early stages of Zn deficiency

Apple trees are extensively cultivated worldwide but are often affected by zinc (Zn) deficiency. Limited knowledge regarding Zn remobilization within fruit crops has hampered the development of efficient strategies for providing adequate amounts of Zn. In the present study, Zn distribution and remobilization were compared among apple trees cultivated under different Zn conditions. Without Zn application, plants showed visible symptoms of Zn deficiency at the shoot tips after 1 year but appeared to grow normally during the first 6 months (early stage of Zn deficiency). Compared with apple plants under sufficient Zn treatment, plants suffering from early‐stage Zn deficiency showed preferential Zn distribution to young leaves and higher Zn levels in phloem, demonstrating that hidden Zn deficiency triggers a highly efficient remobilization of Zn in this species. The in vivo Zn‐nicotianamine complex in phloem tissues, combined with the significant enhanced expression of MdNAS3 and MdYSL6, suggested a positive role for nicotianamine in the phloem remobilization of Zn. These results strongly suggest that a proportion of Zn in the old leaves of apple trees can be efficiently remobilized by phloem transport to the shoot tips, partially in the form of Zn‐nicotianamine, thus protecting apple trees against the early stages of Zn deficiency.     

The effect of dietary molybdenum supplementation on tissue copper concentrations,mohair fibre and carcass characteristics of growing Angora goats

The purpose of this experiment was to better characterize the effects of the interaction between copper (Cu), molybdenum (Mo) and sulphur (S) in the diet on growth, metabolism and fibre characteristics in Angora goats. 15 Angora goats aged 9 months and weighing 21.5 kg on average were used in a ten-week study and allocated to three dietary treatments: Treatment C (10 MJ metabolisable energy, 178 g crude protein, 5.5 mg Cu, 0.57 mg Mo, and 3.4 g S): Treatment M1 (with 7.5 mg Mo) or Treatment M2 (with 15 mg Mo) per animal per day. Dose-dependent increases in the concentrations of Mo (P < 0.01) and Cu (P < 0.05) in plasma were recorded in response to increased dietary intake of Mo. Supplementation of the control diet with increased concentrations of Mo did not produce effects (P > 0.05) on growth rate, feed conversion efficiency, carcass weight or mohair fibre yield and diameter.Haematological status and concentration of Cu in liver and Cu and S in fibre at the end of the study were also not affected (P > 0.05).Concentrations of trichloroacetic acid (TCA) soluble “available” copper in plasma were not significantly different although significant (P < 0.05 and P < 0.01) reductions in the ratio of “available” to total Cu concentrations were observed. This effect was stabilised and maintained after 30 days. It is suggested that the additional Cu in plasma was largely TCA insoluble and possibly in the form of thiomolybdate complexes which may be poorly excreted and not available for uptake to the metabolic sites. It is evident that adequate “available” Cu was present in plasma and that exposure to elevated Mo intake was not severe or long enough to produce clinical symptoms or to affect growth, haematological status or fibre production.     

Distribution and Redistribution of Molybdenum in Black Gram (Vigna mungo L. Hepper) in Relation to Molybdenum Supply

The effect of seven rates of molybdenum (Mo) supply on the distributionand redistribution of Mo in Vigna mungo (black gram) cv. Reguron a Mo-deficient sandy loam was examined from flower bud appearanceto pod set in one experiment and during pod filling to maturityin another. At the three lowest Mo supply rates, N deficiency symptoms typicalof Mo deficiency appeared, and shoot dry matter and shoot nitrogencontent were depressed. Increasing Mo supply increased Mo concentrationsin all plant parts but the response varied with Mo supply andwith plant part. In leaf blades and petioles, Mo concentrationsincreased slightly when the Mo supply increased from severelydeficient to deficient levels but further increases in Mo supplymarkedly increased the Mo concentrations, particularly in immatureand recently matured leaves. In petioles, Mo concentrationsgenerally exceeded those in the blades which they supportedat all levels of Mo supply. At Mo rates greater than that requiredfor maximum growth, Mo concentrations in basal stem segmentsexceeded those in petioles. Molybdenum concentrations in nodulesexceeded those in above ground plant parts except at the highestlevel of Mo supply where the concentrations in basal stem segmentsexceeded those in nodules. In Mo-adequate plants, Mo contents in the trifoliolate leavesdecreased with time suggesting that Mo was readily remobilized.By contrast, in stem segments at all levels of Mo supply, andin trifoliolate leaves in Mo-deficient plants, Mo contents remainedconstant or increased with time suggesting that Mo was not remobilizedin all plant parts or at all levels of Mo supply. Thus, theresults suggest that in black gram Mo was variably mobile, beingphloem immobile at low Mo supply, but phloem-mobile in all plantparts with the possible exception of stem segments at adequateMo supply. The relevance of these results for the developmentof plant tests for Mo deficiency diagnosis is discussed.Copyright1994, 1999 Academic Press Molybdenum, phloem-mobility, redistribution, black gram, Vigna mungo L. Hepper     

Development of three copper metalloenzymes in clover leaves

Subterranean clover (Trifolium subterraneum L. cv Seaton Park) was grown in solution cultures containing adequate nitrogen both with and without Cu. After Cu deficiency had developed, Cu²⁺ was added to some deficient plants and Cu content, protein content, and activities of three Cu metalloenzymes (diamine oxidase [EC1.4.3.6], ascorbate oxidase [EC1.10.3.3] and o-diphenol oxidase [EC1.10.3.1]) were assayed in young and recently matured leaf blades over 11 days during the development of the next three leaves.

Copper deficiency had little effect on protein concentrations, but markedly depressed enzyme activities and Cu concentration in all leaf blades assayed. Within 4 d of adding Cu²⁺ to Cu-deficient plants, Cu concentrations of all the leaf blades increased to adequate values. Enzyme activities only increased to control levels in leaves which had not yet emerged at the time that Cu²⁺ was added.

The results suggest that active holoenzymes of diamine oxidase, ascorbate oxidase, and o-diphenol oxidase can only be synthesized in leaf blades during very early stages of their development.

     

Abscisic Acid Refines the Synthesis of Chloroplast Proteins in Maize (Zea mays) in Response to Drought and Light

To better understand abscisic acid (ABA) regulation of the synthesis of chloroplast proteins in maize (Zea mays L.) in response to drought and light, we compared leaf proteome differences between maize ABA-deficient mutant vp5 and corresponding wild-type Vp5 green and etiolated seedlings exposed to drought stress. Proteins extracted from the leaves of Vp5 and vp5 seedlings were used for two-dimensional electrophoresis (2-DE) and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). After Coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on 2-DE gels. A total of 36 differentially expressed protein spots in response to drought and light were identified using MALDI-TOF MS and their subcellular localization was determined based on the annotation of reviewed accession in UniProt Knowledgebase and the software prediction. As a result, corresponding 13 proteins of the 24 differentially expressed protein spots were definitely localized in chloroplasts and their expression was in an ABA-dependent way, including 6 up-regulated by both drought and light, 5 up-regulated by drought but down-regulated by light, 5 up-regulated by light but down-regulated by drought; 5 proteins down-regulated by drought were mainly those involved in photosynthesis and ATP synthesis. Thus, the results in the present study supported the vital role of ABA in regulating the synthesis of drought- and/or light-induced proteins in maize chloroplasts and would facilitate the functional characterization of ABA-induced chloroplast proteins in C₄ plants.