Plant Growth Regulators and Induction of Leaf Senescence in Nitrogen-Deprived Wheat Plants

The sequence of events and the signals that regulate the remobilization of nitrogen (N) reserves during senescence induced by N starvation were studied in leaf 3, the last fully expanded leaf, in 17-day-old wheat (Triticum aestivum L.) plants. The first event observed was a rapid decrease in the isopentenyl adenosine (iPA) concentration during the first 24 h of N starvation. No differences in t-zeatin riboside and dihydrozeatin riboside concentrations were observed until the end of the assay. During the following 6 days, a decrease in soluble amino acids, chlorophyll, and protein, as well as an increase in soluble sugar concentration and endoproteolytic activity, could be observed. At day 3 of the experiment, the abscisic acid (ABA) concentration in the leaves of N-deprived plants started to increase. After 6 days of N deprivation there was a rise in oxidative stress, as indicated by the increase in malondialdehyde concentration, as well as a decrease in the activities of antioxidant enzymes catalase and ascorbate peroxidase. To analyze interactions with leaf development, the first, second, third, and fourth leaves were studied. iPA concentration decreased in all the leaf stages, including leaf 4, which was not fully expanded. A linear correlation between iPA and protein concentration was determined. These results suggest that the sharp fall in iPA could be the earliest event that induces protein degradation during the development of senescence induced by N deficiency, and that only later is ABA accumulated and oxidative stress developed.     

Photosynthetic CO2 Fixation in the Second Leaf of Wheat Seedlings Grown at Different Conditions of Nitrogen Nutrition

The rates of photosynthetic ₂ assimilation were determined in fully expanded second leaves of 21-day-old wheat (Triticum aestivum L.) seedlings grown on media supplied with nitrate or ammonia and on a nitrogen-free medium (NO₃ ^–- or NH₄ ⁺-treatments and N-deficit treatment, respectively). The maximal quantum efficiency of photosynthesis was independent on conditions of nitrogen nutrition. When leaves were exposed to 0.03% ₂ and high-intensity light, the lowest photosynthetic rate was noted for N-deficit treatment and the highest rate was characteristic of NH₄ ⁺ treatment. The elevation of the ₂ concentration in the gas phase to 0.1% stimulated photosynthesis at high-intensity light in all treatments. The rate of ₂ uptake by the leaf of N-deficient seedlings increased with ₂ concentration to a larger extent than in other treatments and approached the ₂ uptake rate characteristic of the NO₃ ^– treatment. In plants grown on a nitrogen-free medium, the leaf accumulated lesser amounts of reduced nitrogen and higher amounts of starch, but the content of chloroplast protein corresponded to that of NO₃ ^– treatment. In the leaf of NH₄ ⁺-treated seedlings, the rate of ₂ assimilation was higher than in the leaf of NO₃ ^– treated plants, regardless of the composition of the gas mixture. The ammonium-type nutrition, as compared to the nitrate-type nutrition, elevated the amount of reduced nitrogen in the leaf and promoted accumulation of chlorophyll and protein, the chloroplast protein in particular.     

Differences in Sugar Accumulation and Mobilization between Sequential and Non-Sequential Senescence Wheat Cultivars under Natural and Drought Conditions

Wheat leaf non-sequential senescence at the late grain-filling stage involves the early senescence of younger flag leaves compared to that observed in older second leaves. On the other hand, sequential senescence involves leaf senescence that follows an age-related pattern, in which flag leaves are the latest to undergo senescence. The characteristics of sugar metabolism in two sequential senescence cultivars and two non-sequential senescence cultivars under both natural and drought conditions were studied to elucidate the underlying mechanism of drought tolerance in two different senescence modes. The results showed that compared to sequential senescence wheat cultivars, under natural and drought conditions, non-sequential senescence wheat cultivars showed a higher leaf net photosynthetic rate, higher soluble sugar levels in leaves, leaf sheaths, and internodes, higher leaf sucrose synthase (SS) and sucrose phosphate synthase (SPS) activity, and higher grain SS activity, thereby suggesting that non-sequential senescence wheat cultivars had stronger source activity. Spike weight, grain weight per spike, and 100-grain weight of non-sequential senescence cultivars at maturity were significantly higher than those of sequential senescence cultivars under both natural and drought conditions. These findings indicate that the higher rate of accumulation and the higher mobilization of soluble sugar in the leaves, leaf sheaths and internodes of non-sequential senescence cultivars improve grain weight and drought tolerance. At the late grain-filling stage, drought conditions adversely affected leaf chlorophyll content, net photosynthetic rate, soluble sugar and sucrose content, SS and SPS activity, gain SS activity, and weight. This study showed that higher rates of soluble sugar accumulation in the source was one of the reasons of triggering leaf non-sequential senescence, and higher rates of soluble sugar mobilization during leaf non-sequential senescence promoted high and stable wheat yield and drought tolerance.     

Differential Effects of Nitrate and Light on the Expression of Glutamine Synthetases and Ferredoxin-Dependent Glutamate Synthase in Maize

The effects of nitrate and light on the expression of genesfor glutamine synthetase (GS) isoproteins and ferredoxin-dependentglutamate synthase (Fd-GOGAT) were studied in different organsof maize seedlings by analyzing the levels of the respectivepolypeptides and mRNAs. In roots, the levels of plastidic GSand of a novel, root-specific GS molecule localized in the extraplastidiccompartment were increased markedly by nitrate, whereas Fd-GOGATand cytosolic GS remained at their initial levels. Ammonia wasnot effective in inducing the plastidic GS and Fd-GOGAT butit did induce the novel GS isoprotein. In leaves, cytosolicand plastidic GSs and Fd-GOGAT were present in both mesophyllcells (MC) and bundle sheath cells (BSC). Upon addition of nitrate,the level of plastidic GS increased preferentially in MC, andupon exposure of etiolated seedlings to light, the levels ofplastidic GS and Fd-GOGAT increased in BSC in a coordinatedmanner. The relationship between the expression of genes forGSs and Fd-GOGAT and the physiological role of the GS/GOGATcycle is discussed in terms of the characteristics of nitrogenmetabolism in roots, MC, and BSC. (Received August 11, 1992; Accepted September 21, 1992)     

Phosphate Deficiency in Maize. VI. Changes in the Two-Dimensional Electrophoretic Patterns of Soluble Proteins from Second Leaf Blades Associated with Induced Senescence

The effects of P deprivation on the two-dimensional electrophoreticpatterns of soluble proteins were evaluated in maize (Zea maysL.) leaves. P deprivation resulted in decreases in the relativeabundance of 11 of more than 450 polypeptides and increasesin that of 18 polypeptides. These changes are discussed in relationto leaf senescence. (Received March 23, 1995; Accepted June 30, 1995)     

Cloning and Inactivation of Genes Encoding Ferredoxin- and NADH-Dependent Glutamate Synthases in the Cyanobacterium Plectonema boryanum. Imbalances in Nitrogen and Carbon Assimilations Caused by Deficiency of the Ferredoxin-Dependent Enzyme

Glutamate synthase (GOGAT) is a key enzyme in the assimilation of inorganic nitrogen in photosynthetic organisms. We found that, like higher plants, the facultative heterotrophic cyanobacterium Plectonema boryanum had ferredoxin (Fd)- and NADH-dependent GOGATs. The genes glsF, gltB, and gltD were cloned, and structural analyses and target mutageneses demonstrated that glsF encoded Fd-GOGAT and that gltB and gltD encoded the two subunits of NADH-GOGAT. All three mutants lacking one of the GOGAT genes were able to grow photosynthetically and heterotrophically. However, the Fd-GOGAT mutant exhibited a phenotype of marked nitrogen deficiency when grown under conditions of saturating illumination and CO₂ supply. In these conditions the rate of the ammonia uptake from the culture medium was slower in the Fd-GOGAT mutant than in the wild type or in the NADH-GOGAT mutant, but no significant differences were found in the rate of the CO₂ fixation-dependent O₂ evolution among these strains. Our results suggest that, although both Fd- and NADH-GOGATs were operative in the cells growing in light, the contribution of Fd-GOGAT, which directly utilizes photoreducing power for the catalytic reaction, is essential for balancing photosynthetic nitrogen and carbon assimilation.     

A Role for Glutamine Synthetase in the Remobilization of Leaf Nitrogen during Natural Senescence in Rice Leaves

Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of corresponding mRNAs were determined in leaves of hydroponically grown rice (Oryza sativa) plants during natural senescence. The plants were grown in the greenhouse for 105 days at which time the thirteenth leaf was fully expanded. This was counted as zero time for senescence of the twelfth leaf. The twelfth leaf blade on the main stem was analyzed over a time period of −7 days (98 days after germination) to +42 days (147 days after germination). Total GS activity declined to less than a quarter of its initial level during the senescence for 35 days and this decline was mainly caused by a decrease in the amount of GS2 polypeptide. Immunoblotting analyses showed that contents of other chloroplastic enzymes, such as ribulose-1,5-bisphosphate carboxylase/oxygenase and Fd-glutamate synthase, declined in parallel with GS2. In contrast, the GS1 polypeptide remained constant throughout the senescence period. Translatable mRNA for GS1 increased about fourfold during the senescence for 35 days. During senescence, there was a marked decrease in content of glutamate (to about one-sixth of the zero time value); glutamate is the major form of free amino acid in rice leaves. Glutamine, the major transported amino acid, increased about threefold compared to the early phase of the harvest in the senescing rice leaf blades. These observations suggest that GS1 in senescing leaf blades is responsible for the synthesis of glutamine, which is then transferred to the growing tissues in rice plants.     

Morphophysiological Indices of the Source Leaf in Wheat Plants Acclimated to Conditions of Nitrogen Nutrition

Growth, leaf and cell morphology, and the chemical composition of the second leaf were studied in wheat (Triticum aestivumL., cv. Inna) plants grown on the medium containing nitrate, ammonium, or no nitrogen at all. Independent of the nitrogen nutrition, the second leaf of the 21-day-old plants matures and functions as a source of assimilates. Both ammonium nutrition and nitrogen deficiency decreased the fresh weight, area, and cell size in the leaf; however, the conditions of nitrogen nutrition did not affect the dry weight of the leaf. Nitrogen starvation increased and ammonium nutrition decreased the relative content of the cell walls in the dry weight. In the nitrate-fed plants, the leaf content of sucrose increased, and the contents of reduced nitrogen (N_red) and protein were lower than in the ammonium treatment. Reciprocally, the contents of reduced nitrogen and protein were highest in the ammonium treatment, the content of sucrose was lowest, with starch practically absent from the leaf. The nitrogen-starved leaf accumulated a large amount of starch, the N_redcontent was two times lower than in the ammonium-fed plants, and the protein content was similar to that in the nitrate-fed plants. Thus, leaf and cell morphology and the content of N_red, protein, and carbohydrate changes in different ways during wheat acclimation to the condition of nitrogen nutrition. By assessing the cell wall weight, the authors established that, depending on nitrogen nutrition, this cell compartment accepts a variable flow of carbon.     

Assimilation of Nitrogen in Mutants Lacking Enzymes of the Glutamate Synthase Cycle

¹⁵N labelling was used to investigate the pathway of nitrogenassimilation in photorespiratory mutants of barley (Hordeumvulgare cv. Maris Mink), in which the leaves have low levelsof glutamine synthetase (GS) or glutamate synthase, key enzymesof ammonia assimilation. These plants grew normally when maintainedin high CO₂, but the deletions were lethal when photorespirationwas initiated by transfer to air. Enzyme levels in roots weremuch less affected, compared to leaves, and assimilation oflabelled nitrate into amino acids of the root showed very littledifference between wild type and mutants. Organic nitrogen wasexported from roots in the xylem sap mainly as glutamine, levelsof which were somewhat reduced in the GS-deficient mutant andenhanced in the glutamate synthase deficient mutant. In theleaf, the major effect was seen in the glutamatesynthase mutant,which had an extremely limited capacity to utilize the importedglutamine and amino acid synthesis was greatlyrestricted. Thiswas confirmed by the supply of [¹⁵N]-glutamine directly to leaves.Leaves of the GS-deficient mutant assimilatedammonia at about75% the rate found for the wild type, and this was almost completelyeliminated by addition of the inhibitormethionine sulphoximine.Root enzymes, together with residual levels of the deleted enzymesin the leaves, have sufficient capacityfor ammonia assimilation,through the glutamate synthase cycle, to provide adequate inputof nitrogen for normal growth of themutants, if photorespiratoryammonia production is suppressed. Key words: Hordeum vulgare, ¹⁵N, glutamine synthetase, glutamate synthase, ammonia assimilation     

冬小麦叶片持绿能力及其衰老特征研究

以12个冬小麦(Triticum aestivum L.)品种为供试材料,通过田间实验连续2年于开花后定期测定各品种的绿叶数目、绿叶面积、叶绿素和MDA含量以及SOD和CAT活性等指标,并以生理成熟时的保绿度、衰老启动时间为指标进行Hierarchical聚类分析,对小麦品种持绿能力进行分级.结果表明,参试冬小麦品种可分为持绿和非持绿两种类型,‘潍麦8号'(WM8)和‘豫麦66'(YM66)两年均表现为持绿型小麦.在整个灌浆期,持绿型小麦品种绿叶数目、面积、叶绿素含量明显高于非持绿型品种,叶片保护酶SOD与CAT活性也较非持绿小麦强,而其MDA含量明显低于非持绿型小麦品种.持绿型小麦叶片衰老启动时间延迟,生育后期绿叶面积较大,光合作用时间延长,具有较高的产量.本研究结果为冬小麦的品种选育、布局等相关研究奠定基础.     

Tissue Distribution of Glutamate Synthase and Glutamine Synthetase in Rice Leaves : Occurrence of NADH-Dependent Glutamate Synthase Protein and Activity in the Unexpanded, Nongreen Leaf Blades

To further explore the function of NADH-dependent glutamate synthase (GOGAT), the tissue distribution of NADH-GOGAT protein and activity was investigated in rice (Oryza sativa L.) leaves. The distributions of ferredoxin (Fd)-dependent GOGAT, plastidic glutamine synthetase, and cytosolic glutamine synthetase proteins were also determined in the same tissues. High levels of NADH-GOGAT protein (33.1 μg protein/g fresh weight) and activity were detected in the 10th leaf blade before emergence. The unexpanded, nongreen portion of the 9th leaf blade contained more than 50% of the NADH-GOGAT protein and activity per gram fresh weight when compared with the 10th leaf. The expanding, green portion of the 9th leaf blade outside of the sheath contained a slightly lower abundance of NADH-GOGAT protein than the nongreen portion of the 9th blade on a fresh weight basis. The fully expanded leaf blades at positions lower than the 9th leaf had decreased NADH-GOGAT levels as a function of increasing age, and the oldest, 5th blade contained only 4% of the NADH-GOGAT protein compared with the youngest 10th leaf blade. Fd-GOGAT protein, on the other hand, was the major form of GOGAT in the green tissues, and the highest amount of Fd-GOGAT protein (111 μg protein/g fresh weight) was detected in the 7th leaf blade. In the nongreen 10th leaf blade, the content of Fd-GOGAT protein was approximately 7% of that found in the 7th leaf blade. In addition, the content of NADH-GOGAT protein in the 10th leaf blade was about 4 times higher than that of Fd-GOGAT protein. The content of plastidic glutamine synthetase polypeptide was also the highest in the 7th leaf blade (429 μg/g fresh weight) and lowest in nongreen blades and sheaths. On the other hand, the relative abundance of the cytosolic glutamine synthetase polypeptide was the highest in the oldest leaf blade, decreasing to 10 to 20% of that value in young, nongreen leaves. These results suggest that NADH-GOGAT is important for the synthesis of glutamate from the glutamine that is transported from senescing source tissues through the phloem in the nongreen sink tissues in rice leaves.     

Changes in the Number and Size of Chloroplasts during Senescence of Primary Leaves of Wheat Grown under Different Conditions

Changes in the number and size of chloroplasts in mesophyllcells were investigated in primary leaves of wheat from fullexpansion to yellowing under different growth conditions. Thenumber of chloroplasts per cell decreased slowly, although thedecrease was steady and statistically significant, until thelast stage of leaf senescence, when rapid degradation of chloroplaststook place. Rates of leaf senescence, or the decline in thenumber of chloroplasts, varied greatly among plants grown atdifferent seasons of the year, but about 20% of chloroplastsalways disappeared during the phase when steady loss of chloroplastsoccurred. The area of chloroplast disks also decreased graduallybut significantly, with a rapid decrease late in senescence.Thus, the total quantity of chloroplasts per mesophyll celldecreased substantially during leaf senescence. Yellowed leavescontained numerous structures that resemble oil drops but nochloroplasts. Decreases in rates of photosynthesis that occurduring senescence may, therefore, be largely due to decreasesin the quantity of chloroplasts. However, a better correlationwas found between the decrease in the maximum capacity for photosynthesisand the degradation of RuBP carboxylase. When plants had beengrown with a sufficient supply of nutrients, the number of chloroplastsdecreased steadily but at a reduced rate and the reduction inthe area of chloroplast disks was strongly suppressed. Thus,the quantitative decrease in chloroplasts in senescing leavesappears to be regulated by the requirements for nutrients (nitrogen)of other part of the plant. ³Present address: Department of Biology, Faculty of Science,Toho University, Miyama, Funabashi, Chiba, 274 Japan     

Effect of Stress Conditions Induced by Temperature, Water and Rain on Senescence Development

The effect of different stress conditions was studied in segmentsof oat leaves (Avena sativa L. cv. Suregrein). Temperature and water (water dificit and rain) stress conditionsaccelerated senescence development. Any stress condition leadingto an increase in membrane permeability accelerated photooxidativephenomena in light, and senescence development including chlorophyllbreakdown and hydroperoxides increase. On the contrary, in darkness,any stress condition prevented chlorophyll breakdown and senescencedevelopment except for proteolysis, despite an increase in permeability. Temperature stress did not increase proteolysis. Water stressinduced in a humid chamber increased proteolysis, but it didnot increase proteolysis when water stress was induced by floatingin osmotic solution. (Received November 17, 1986; Accepted August 19, 1987)     

Productivity and Growth of a Natural Population of the Smallest Free-Living Eukaryote under Nitrogen Deficiency and Sufficiency

The influence of dissolved inorganic nitrogen (DIN) enrichments on cell-normalized carbon uptake rate, chlorophyll a content, and apparent cell size of a picoeukaryote (<1 m) (Ostreococcus tauri, the smallest eukaryotic cell) from a natural summer phytoplanktonic assemblage (<200 m) in a northern Mediterranean Lagoon (Thau Lagoon) was studied in 20-L enclosures in June 1995. The natural planktonic community was incubated in situ for 24 h with initial ammonium and nitrate enrichments and compared to a control without enrichment. O. tauri cell-normalized productivity was estimated from the combination of flow cytometric (FCM) enumeration and 2-h (radioactive) carbonate incorporation measured on post-incubation size fractions (<1m). No difference between the effects of the two DIN sources of enrichment on the studied biological parameters was measured during this experiment. Growth of natural O. tauri was perturbed by the low DIN availability in the control with drastic changes in cell productivity, chlorophyll content, and cell cycle (from the variations in apparent cell size) as compared to the DIN sufficiency conditions. On the other hand, a very high specific growth rate for natural O. tauri, up to 8 day^–1 under DIN enrichments, has been estimated from production and abundance data obtained during this experiment. This supports values measured in culture and suggests that the yearly high contribution of picophytoplankton to the total primary production in Thau Lagoon is likely to be due to their high growth rate rather than the previously suggested lack of grazing pressure.     

Leaf Senescence Induced by Mild Water Deficit Follows the Same Sequence of Macroscopic, Biochemical, and Molecular Events as Monocarpic Senescence in Pea

We have compared the time course of leaf senescence in pea (Pisum sativum L. cv Messire) plants subjected to a mild water deficit to that of monocarpic senescence in leaves of three different ages in well-watered plants and to that of plants in which leaf senescence was delayed by flower excision. The mild water deficit (with photosynthesis rate maintained at appreciable levels) sped up senescence by 15 d (200 degrees Cd), whereas flower excision delayed it by 17 d (270 degrees Cd) compared with leaves of the same age in well-watered plants. The range of life spans in leaves of different ages in control plants was 25 d (340 degrees Cd). In all cases, the first detected event was an increase in the mRNA encoding a cysteine-proteinase homologous to Arabidopsis SAG2. This happened while the photosynthesis rate and the chlorophyll and protein contents were still high. The 2-fold variability in life span of the studied leaves was closely linked to the duration from leaf unfolding to the beginning of accumulation of this mRNA. In contrast, the duration of the subsequent phases was essentially conserved in all studied cases, except in plants with excised flowers, where the degradation processes were slower. These results suggest that senescence in water-deficient plants was triggered by an early signal occurring while leaf photosynthesis was still active, followed by a program similar to that of monocarpic senescence. They also suggest that reproductive development plays a crucial role in the triggering of senescence.     

Involvement of the Phospholipid Sterol Acyltransferase1 in Plant Sterol Homeostasis and Leaf Senescence

Genes encoding sterol ester-forming enzymes were recently identified in the Arabidopsis (Arabidopsis thaliana) genome. One belongs to a family of six members presenting homologies with the mammalian Lecithin Cholesterol Acyltransferases. The other one belongs to the superfamily of Membrane-Bound O-Acyltransferases. The physiological functions of these genes, Phospholipid Sterol Acyltransferase1 (PSAT1) and Acyl-CoA Sterol Acyltransferase1 (ASAT1), respectively, were investigated using Arabidopsis mutants. Sterol ester content decreased in leaves of all mutants and was strongly reduced in seeds from plants carrying a PSAT1-deficient mutation. The amount of sterol esters in flowers was very close to that of the wild type for all lines studied. This indicated further functional redundancy of sterol acylation in Arabidopsis. We performed feeding experiments in which we supplied sterol precursors to psat1-1, psat1-2, and asat1-1 mutants. This triggered the accumulation of sterol esters (stored in cytosolic lipid droplets) in the wild type and the asat1-1 lines but not in the psat1-1 and psat1-2 lines, indicating a major contribution of the PSAT1 in maintaining free sterol homeostasis in plant cell membranes. A clear biological effect associated with the lack of sterol ester formation in the psat1-1 and psat1-2 mutants was an early leaf senescence phenotype. Double mutants lacking PSAT1 and ASAT1 had identical phenotypes to psat1 mutants. The results presented here suggest that PSAT1 plays a role in lipid catabolism as part of the intracellular processes at play in the maintenance of leaf viability during developmental aging.Sterols are components of most eukaryotic membranes; as such, they are important regulators of membrane fluidity and thus influence membrane properties, functions, and structure (Demel and De Kruyff, 1976; Bloch, 1983; Schuler et al., 1991; Roche et al., 2008). Unlike animals, in which cholesterol is most often the unique end product of sterol biosynthesis, each plant species has its own distribution of sterols, with the three most common phytosterols being sitosterol, stigmasterol, and campesterol (Benveniste, 2004). In addition to their free sterol form, phytosterols are also found as conjugates, particularly fatty acyl sterol esters (SE). Since SE are hardly integrated into the bilayer of the membranes (Hamilton and Small, 1982), the biochemical process of sterol acylation is believed to play a crucial role in maintaining free sterol concentration in the cell membranes (Lewis et al., 1987; Dyas and Goad, 1993; Chang et al., 1997; Sturley, 1997; Schaller, 2004). In other words, SE are generally thought to constitute a storage pool of sterols when those are present in amounts greater than immediately required for the cells. For instance, in plants, accumulation of SE has been described during seed maturation and senescence or when plant cell cultures reach stationary phase (Dyas and Goad, 1993, and refs. therein) as well as in mutant lines overproducing sterols (Gondet et al., 1994; Schaller et al., 1995).In mammals and yeast, the genes involved in sterol esterification have been known for a long time. These genes encode two types of enzymes responsible for the formation of SE in animals, the Acyl-Coenzyme A:Cholesterol Acyltransferase (ACAT) and the Lecithin:Cholesterol Acyltransferase (LCAT). ACAT, which catalyzes an acyl-CoA-dependent acylation, is a membrane-bound enzyme acting inside the cells (Chang et al., 1997). LCAT, which is evolutionarily unrelated to ACAT, catalyzes transacylation of acyl groups from phospholipids to sterols. It is a soluble enzyme present in the blood stream, where it is an important regulator of circulating cholesterol (Jonas, 2000). The budding yeast Saccharomyces cerevisiae has two enzymes of the ACAT type for the synthesis of SE (Yang et al., 1996).In plants, genes encoding enzymes responsible for SE formation have long been unknown. Based on biochemical studies, it was suggested that phospholipids and/or neutral lipids could serve as acyl donors (Garcia and Mudd, 1978a, 1978b; Zimowski and Wojciechowski, 1981a, 1981b). The identification in the Arabidopsis (Arabidopsis thaliana) genome of two genes involved in sterol esterification was based on homology searches. First, the phospholipid:sterol acyltransferase gene AtPSAT1 (At1g04010) was found to display consistent identity with the mammalian LCAT and then was biochemically characterized by expression in Arabidopsis (Noiriel, 2004; Banas et al., 2005). The encoded protein was shown to be associated with microsomal membranes and to catalyze the transfer of unsaturated fatty acyl groups from position sn-2 of phosphatidylethanolamine (and phosphatidylcholine to a lesser extent) to sterols. The preferred acceptor molecules of PSAT1 were cholesterol, a minor biosynthetic end product in Arabidopsis, then campesterol and sitosterol, the two main end products. Sterol coincubation studies performed with this microsomal enzymatic assay showed that sterol precursors such as cycloartenol or obtusifoliol, which were poor substrates when incubated alone, were preferentially acylated in the presence of sitosterol, suggesting an activation of the enzyme by sitosterol (Banas et al., 2005). Another sterol acyltransferase gene, AtASAT1 (At3g51970), was identified in a survey of members of the Arabidopsis superfamily of membrane-bound O-acyltransferases with a yeast ACAT mutant functional complementation approach (Chen et al., 2007). AtASAT1 encodes a protein structurally related to the animal and yeast ACATs. This enzyme was shown to prefer saturated fatty acyl-CoAs as acyl donors and cycloartenol as the acyl acceptor. Overexpression of AtASAT1 in seeds of Arabidopsis resulted in a strong accumulation of cycloartenol fatty acyl esters accompanied by an increase of the whole SE content of these seeds and, in spite of a slight decrease of the free sterol pool, an increase of the total sterol content of the transgenic seeds by up to 60% compared with that of the wild type (Chen et al., 2007). We took advantage of the availability of Arabidopsis T-DNA insertion mutants of these two genes to investigate their respective physiological roles. Here, we report on the involvement of AtPSAT1 in leaf senescence, its major contribution to SE formation in leaves and seeds, and also its essential role in free sterol homeostasis in these organs.     

Response of Glutamine Synthetase and Glutamate Synthase Isoforms to Nitrogen Sources in Rice Cell Cultures

As a model system with no photorespiration and no long distancetransport, rice cell cultures (Oryza saliva L. cv Sasanishiki)were used to investigate the effect of nitrogen sources on thelevels of isoforms of glutamine synthetase (GS) and glutamatesynthase (GOGAT). Isoforms of GS and GOGAT were analyzed byimmunoblotting methods and their activities in early growthphase of the cells. Cytosolic type GS (41 kDa subunit) and NADH-GOGATwere the major isoforms in the rice cells grown in normal R-2medium. However, contents of plastid type GS (44 kDa subunit)and Fd-GOGAT increased in response to NO_–³ supply. NADH-GOGATactivity also increased following the supply of NO_–³.In vitro translated products from poly(A)⁺RNA prepared fromthe cells showed that the precursor of plastid type GS (49 kDa)was detected at 48 h after the inoculation. Supply of NH₊⁴ resultedin an increase in NADH-GOGAT activity but had no effect on thelevels of Fd-GOGAT, of polypeptides of the plastid type GS orof the corresponding mRNAs. (Received May 30, 1990; Accepted August 23, 1990)     

Leaf Regulation of the Nitrogen Concentration in the Grain of Wheat Plants

The regulation of the final grain N concentration in wheat (Triticumaestivum) plants was studied through the alteration of the source/sinkratio. Plants were grown in a greenhouse in pots with soil,and fertilized with a supraoptimal N supply. The plants were divided into six groups. In one treatment, plantsremained untouched as a control (Treatment 1). In another group,all the ears except that of the main tiller were removed atflowering (Treatment 2). All other plants were de-tillered afterthe emergence of the third leaf, leaving only one tiller perplant. At flowering, one plant set was left untouched (Treatment3). In a second group, all the leaves were excised (Treatment4). In another group half the spikelets of the ear were excised(Treatment 5) and in the last group three-quarters of the spikeletswere excised (Treatment 6). Ear excision produced an increase in individual grain weightand the grain N concentration above the normal N concentrationobserved in this cultivar. The final N concentration was correlatedwith the concentration of free amino acids in the flag leaf34 d after flowering. It is concluded that in intact plants grain protein synthesisis substrate-limited by the amino acid export pool in the leaves,and grain excision increases the availability of amino acidsto be transported to the remaining grains. Key words: Amino acids, grain N concentration, nitrogen, remobilization, wheat     

The Significance of Vascular Connection in Regulating Senescence of the Detached Flag Leaf of Wheat

The relationship between the accumulation of soluble substancesand the senescence of a detached flag leaf of wheat in the lightwas investigated. With respect to transport to and from theleaf, a detached flag leaf can be considered as either an ‘open’or a ‘closed’ system. A closed system was obtainedby cutting the flag leaf at the base of the leaf sheath, thussevering the vascular connection between the leaf and the stem.An open system was prepared by excising the wheat stem, belowthe flag leaf insertion node, thereby preserving vascular connectionsbetween the flag leaf, stem and ear. By varying the number ofnodes left on the stem, or having the ear either intact or detached,soluble carbohydrates and soluble nitrogenous compounds wereinduced to accumulate within the leaf blade at different rates.Treatments which restricted transport of carbohydrate and nitrogen,out of the detached leaf were found to promote senescence. Senescencewas most rapid when the leaf system was ‘closed’and was considerably delayed when the system was ‘open’and the ear intact. The onset of senescence was closely associatedwith the attainment of a threshold concentration of ethanol-solublecarbohydrate in the leaf, while the rate of senescence was modifiedby the number of attached nodes or by exogenous cytokinin treatments. Key words: Wheat, Flag leaf, Senescence