Journal of Experimental Botany, Vol. 52, No. 362, pp. 1761-1768,
September 1, 2001
© 2001 Oxford University Press
Original Papers |
The export of amino acid in the phloem is altered in wheat plants lacking the short arm of chromosome 7B
IBYF-CONICET, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453 (1417), Buenos Aires, Argentina
Received 5 January 2001; Accepted 24 May 2001
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Abstract |
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Grain protein content is one of the major determinants of the baking and nutritional quality of wheat. It has previously been reported that the ditelosomic line of wheat (Triticum aestivum L.) CSDT7BL, where the short arm of chromosome 7B is missing, shows a lower grain protein concentration than the normal line, but a similar grain yield. In the present paper the growth and nitrogen (N) metabolism of wheat plants cv. Chinese Spring (CS) and its ditelosomic line CSDT7BL were compared. When plants were grown to maturity in pots with different N supplements, the wild-type line showed a higher grain protein concentration and a lower straw N concentration than the ditelosomic line at every N level analysed, suggesting a deficiency in the N remobilization capacity. When 15-d-old plants were grown in a growth cabinet in pots with sand, and supplied with nutrient solutions of different nitrate concentrations, the ditelosomic line showed no differences in N uptake per unit of root dry weight, nitrate reductase activity, nitrate, total N concentration or free amino acid concentration. However, the ditelosomic line showed a decreased capacity to export amino acids in the phloem under high N, independently of the N source. This deficiency was also observed under dark-induced senescence. The diminished export of amino acids to the phloem was principally caused by a decrease in the export of Glu, Asp, and Gln. It is suggested that the decrease in grain protein concentration in the ditelosomic line is a consequence of defective export in the phloem of these amino acids.
Key words: Triticum aestivum, phloem, N metabolism, amino acids.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Grain protein content is one of the major determinants of the baking and nutritional quality of wheat. Although the location of most of the wheat genes encoding the mayor grain proteins has been established (Müller et al., 1995
), the physiology and the genetic control of total grain protein content is still far from clear. Most of the genes involved in the regulation of protein concentration are believed to have a minor effect on this character, and are difficult to identify because of the masking effect of the environment. As a consequence, most of the wheat chromosomes and chromosome groups have been reported as affecting grain protein concentration (Konzak, 1977). A bimodal or trimodal frequency distribution for protein content was observed among progenies, suggesting the existence of more than one important gene for nitrogen (N) concentration in bread wheat (Beniani and Busch, 1992). A gene for high protein content on chromosome 6 of T. turgidum has been located (Joppa and Cantrell, 1990), but no single gene for grain protein concentration in bread wheat has been characterized.
Mutant lines for different genes have been extremely useful in the study of the regulation of metabolic pathways in several plant species. However, in hexaploid wheat the detection of single-gene mutant lines is unlikely due to the similarity of the homologous genomes present, where the effect of a mutation in one chromosome can remain hidden by the expression of homologous genes. As an alternative, ditelosomic lines, where a whole chromosome arm is missing, can be useful tools for studying the effects of the lack of genetic information on plant characters of wheat. The ditelosomic lines of wheat cv. Chinese Spring (CS) have frequently been used for genetic studies of grain protein concentration (Konzak, 1977). It has been reported that many of these lines show an increased grain protein concentration. These increases were negatively correlated with grain yield, indicating that the absence of the chromosome arm did not increase the protein content (Law and Brown, 1977). However, in experiments with several ditelosomic lines of wheat cv. Chinese Spring it was observed that the ditelosomic line CSDT7BL, where the short arm of chromosome 7B is missing, shows a decrease in grain protein concentration (15–20%) compared with the euploid line, in spite of a slightly lower grain yield (Barneix et al., 1998). Further experiments with field-grown plants indicated that the reduced protein concentration in the grains of CSDT7BL could be due to a deficient N remobilization under high N supply. At maturity the ditelosomic line retained a higher N concentration in the straw (Fatta et al., 2000), suggesting that the short arm of chromosome 7B of cv. Chinese Spring carries important information involved in the remobilization of nitrogenous compounds from leaves into grains (Barneix et al., 1998; Fatta et al., 2000).
In the present paper, growth and N metabolism were compared in wheat plants cv. Chinese Spring and its ditelosomic line CSDT7BL. Plants grown to maturity under different N supplements were analysed and N utilization by young plants of both lines exposed to different growth conditions which alter N metabolism and transport was studied.
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Materials and methods |
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Plant materials
Seeds of the euploid (CS) and the 7BL ditelosomic line (CSDT7BL) of wheat (Triticum aestivum L. cv. Chinese Spring), were supplied by the Cereal Research Department, John Innes Centre, UK, and multiplied in the field.
Plant culture
Greenhouse experiment:
Seeds were germinated on moist filter paper at 25 °C in the dark. Forty-eight hours later five seedlings were transplanted to each 4 kg plastic pot containing homogenized topsoil. Plants were watered daily. At emergence and at booting 2, 3, 4 g or no urea was applied per pot, with five replicates per treatment, where each replicate was represented by one pot. At maturity plants were harvested, separated into grain and straw, weighed and analysed for total N as indicated below.
Growth cabinet experiments:
Seeds were germinated on moist filter paper at 25 °C in the dark. Forty-eight hours later five seedlings were transplanted to a 300 cm3 plastic pot containing washed sand. Plants were grown in a growth cabinet with a constant temperature of 25 °C, a photoperiod of 16 h and a photosynthetic photon flux density of 300 µmol m-2 s-1 provided by General Electric® 400 W HPL lamps.
Each pot was supplied with 50 ml d-1 nutrient solution (Hoagland and Arnon, 1950) modified as indicated in each experiment. All experiments consisted of five replicates, where each replicate was a pot containing five plants. For individual amino acid analysis, three replicates were used where two pots (10 plants) represented a single sample; this was done in order to get enough phloem exudate.
Experimental design
Plant growth under different 
supplies (Experiment 1):
Pots were divided into four groups, each one receiving nutrient solution containing, 1.0, 5.0, 10.0 or 15.0 mM KNO3. After 15 d plants were sampled, phloem exudate was collected as indicated below, and plants were harvested and stored for further analyses.
Plant growth under different 
supplies (Experiment 2):
Pots were divided into two groups, each one receiving 1.0 or 5.0 mM (NH4)2PO4, for 15 d. Plants received the same treatment as in Experiment 1.
Effects of N starvation on plant growth and N mobilization (Experiment 3):
The plants were fertilized with a nutrient solution containing 15.0 mM KNO3 for 12 d after transplantation. From then on (day 0), the plants were supplied with a nitrogen-free solution. At different times, phloem exudate was collected and plants were harvested as indicated below.
Effects of induced senescence on plant growth and N mobilization (Experiment 4):
15-d-old plants grown for 13 d with 10.0 mM , were placed in continuous dark. Samples were taken at 0, 24, 48, and 72 h. Phloem exudate was collected as indicated below, and leaves were harvested and stored for further analyses.
Amino acid composition of the phloem exudate (Experiment 5):
Plants were cultivated for 15 d with nutrient solution containing 15 mM . The two fully expanded leaves of each plant were collected in order to analyse the individual amino acid concentrations in the leaf tissue and in the phloem exudate solution.
Phloem exudate collection
The two youngest fully expanded leaves of each plant were excised, and the cut ends of ten leaves (20 leaves were used in order to obtain adequate amounts of amino acids) were placed into 2 ml of 20 mM ethylene-diamine-tetraacetic acid (EDTA) (pH 6.0) solution (Urquhart and Joy, 1981), in 40 ml glass tubes. After 15 min in the dark, this preincubation solution was discarded since the exudate primarily consists of xylem or cellular fluid. The leaves were rinsed, transferred to another 2 ml of the same solution, and kept for 3 h in the dark to avoid transpiration. At the end of the incubation time, 300 mg of tissue was harvested and used for protein analysis, as described below. The remaining leaf tissue was freeze-dried for further analysis, and the exudate solution was frozen at -18 °C. Previous experiments showed that there were no changes in the concentration of leaf amino acids during the 3 h dark period. The remaining part of the shoot was harvested and dried at 60 °C for 48 h.
Nitrate reductase activity
At sampling time fully expanded leaves were excised and used for the assay of ‘in vivo’ nitrate reductase (NR, EC 1.6.6.1) activity as described earlier (Jaworsky, 1971).
Plant analysis
The amino acids were extracted overnight at -18 °C from 50 mg of the freeze-dried leaf tissue, with 5 ml of a mixture of chloroform/ethanol/water (5:12:3 by vol.) (Barneix et al., 1984). Total free amino acids in the leaf extracts and in the phloem exudate were analysed with the ninhydrin reagent (Yemm and Cocking, 1955). The concentration of the individual amino acids from phloem exudate and leaf extracts were analysed in a Waters® HPLC with a Waters PICOTAG® column after ultrafiltration and derivation as described in the Waters PICOTAG® method.
Soluble sugars were extracted from 50 mg of the freeze-dried tissue in 5 ml of water for 10 min at 100 °C. Sugars in the leaf extracts and in phloem exudate were analysed with the anthrone reagent (Yemm and Willis, 1954).
Total nitrogen was analysed with a modified Kjeldahl procedure after digestion in concentrated H2SO4 (Barneix et al., 1992). Tissue was extracted using 25 mg dry tissue in 10 ml of water for 90 min at 30 °C, and analysed with a sulphuric–salicylic acid mixture (Cataldo et al., 1975). Total soluble protein in the leaf tissue was extracted from 300 mg fresh leaf tissue with 3 ml of 25 mM phosphate buffer (pH 7.0) that had been ground in a mortar and centrifuged for 10 min at 157 000 g. The soluble protein concentration in the supernatant was determined by the Lowry method (Lowry et al., 1951). N uptake per unit of root dry weight was calculated dividing the ratio of total plant N content by the root dry weight.
Statistical treatment
ANOVA and regression analyses of the data were performed with Statgraphics (R) software.
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Results |
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Greenhouse experiment
Plants of Chinese Spring wheat and the ditelosomic line CSDT7BL grown to maturity in pots with soil with different N additions showed no differences in grain yield (data not shown). However, the normal line showed a higher protein concentration than the ditelosomic line at every nitrogen treatment (Fig. 1A
). The N concentration in the straw, on the other hand, was higher in the ditelosomic line in every treatment, except at the highest N addition, where both lines showed the same N concentration (Fig. 1B).
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) CSDT7BL and (
) CS. Bars represent ±SE (n=5).
Growth cabinet experiments
Plant growth under different supplies (Experiment 1):
When plants of both lines were grown for 15 d with nutrient solutions of different N concentrations, the plants of the wild type produced more biomass than the ditelosomic line in every treatment (Fig. 2A). Only the 1.0 mM concentration was limiting for growth.
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The total N content of the plants showed a large increase between 1.0 to 5.0 mM
in both lines. However, while no further increment was observed in the CSDT7BL, the increase continued in the wild type, and the N content of the CS was higher than in the ditelosomic line at all concentration (Fig. 2B
). When expressed per unit dry weight the two lines possessed similar N concentration (Fig. 2C). The N uptake per unit of root dry weight increased when nitrate supply increased from 1.0 to 5.0 mM, but showed no differences between genotypes (data not shown). The concentration in the leaf tissue remained low in both lines until the nitrate concentration of the nutrient solution was 5.0 mM, after which it increased in a similar way in both genotypes (Fig. 2D).
The soluble protein concentration in the fully expanded leaves (used for obtaining phloem exudate) showed no difference between lines, and both lines also showed the same nitrate reductase activity that increased with a higher N supply (data not shown). Likewise there were no differences in the total amount of leaf free amino acids (Fig. 3A). However, the amino acid concentration in the phloem exudate of CS was higher than in CSDT7BL and the difference increased with the N concentration (Fig. 3B). The leaf sugar concentration was high in the N-deficient plants, and decreased when enough N was supplied (Fig. 3C), while the sugar concentration in the phloem exudate was the same in both lines and for all N treatments (Fig. 3D).
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Plant growth under different supplies (Experiment 2):
When plants were supplied for 15 d with as the only N source, the amino acid concentration in the leaf tissue increased with the concentration in both lines (Fig. 4A). The amino acid concentration in the phloem exudates for the plants fed 5 mM was lower in the CSDT7BL plants than in the normal ones (Fig. 4B). Thus the CSDT7BL plants in Experiments 1 and 2, showed a lower amino acid concentration in the phloem exudate with both N sources.
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Effects of N starvation on plant growth and N mobilization (Experiment 3):
When plants grown for 12 d with 15.0 mM were deprived of N supply, shoot growth continued for a further 5 d in both lines, without any change in the total N content (Fig. 5A, B). As a consequence, there was a continued decrease in total N concentration in both lines, and the tissue concentration decreased during the first 2 d and then remained constant at a low level in the two lines (data not shown). The leaf amino acid concentration, on the other hand, decreased in both lines until the end of the 5 d experiment with a lower concentration in the CS (Fig. 5C). The amino acid concentration in the phloem exudate decreased over the 5 d as well, although during the first 2 d of N starvation the decline was faster in the CSDT7BL plants (Fig. 5D). The concentration of soluble sugars in the leaves and in the phloem exudate of both lines was hardly affected by withdrawal (data not shown). Consequently, the amino acid/sugar ratio in the phloem exudate of CSDT7BL fell from the start of N starvation, with a slower decline in the CS line (data not shown).
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Effects of induced senescence on plant growth and N mobilization (Experiment 4):
When the light supply to 15-d-old plants was interrupted for 72 h, the free amino acid concentration in the leaf tissue increased more than 3-fold in both lines (Fig. 6A). The phloem exudate amino acid concentration of the CS plants increased more than 2-fold during the 72 h dark period, while in the CSDT7BL they remained unaltered during this period (Fig. 6B). This is in agreement with the results obtained in experiments 1 and 2.
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Amino acid composition of the phloem exudate (Experiment 5):
The total concentration of the individual amino acids in leaves of plants grown for 15 d with 15 mM , was the same for CS and CSDT7BL plants. However, the ditelosomic line showed a higher Ser and a lower Asn concentration than the normal line. Asp, Ser, Glu, Thr, Ala, and Gln were the leaf most abundant amino acids in both lines, accounting for 90% of the total (Table 1).
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In the phloem exudate, on the other hand, there were major differences in the total amino acid concentration. This decrease resulted primarily in a decline in the concentration of amino acids: Glu was 52.3%, Asp 49.2%, Gln 50.5%, Tyr 35.4%, Thr 30.5%, Phe 100%, Ile 58.8%, Val 43.7%, and Ala 24.3% lower, while the rest of the determined amino acids was not affected (Table 1
).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Wheat is an allohexaploid, with three homologous chromosome groups. In a ditelosomic line the absence of a chromosome arm of a pair of homologous chromosomes may have some impact on particular characteristics. In N-fertilized field experiments (Barneix et al., 1998
; Fatta et al., 2000), as well as that conducted in the greenhouse, the ditelosomic line CSDT7BL showed little difference in plant weight and grain yield with the normal line. However, in the present experiments, during the early state of growth, the ditelosomic line had a 40% lower weight than the euploid line (Fig. 2A).
The results of the experiment with plants grown to maturity in pots with soil are in agreement with the previous results observed in the field (Fatta et al., 2000). The CSDT7BL showed a lower grain protein concentration and a higher straw N concentration than CS, in spite of the similar growth observed. On the other hand, in the growth cabinet experiments, both lines showed the same N uptake per unit of root dry weight, NR activity, and similar total N and concentration. (Fig. 2C, D). However, CS plants showed increased plant growth and total N content at high supply (Fig. 2A, B). The concentration of amino acids in the phloem was quite different, as revealed by the EDTA technique. In the CSDT7BL plants the amino acid concentration was unaffected, despite the increasing supply, and remained at the same level as in the 5.0 mM -fed CS plants. The rate of amino acid export in the phloem in the CS plants was highly dependent on N supply (Fig. 3B) although sugar export from the phloem remained constant in both lines at all N supplied, indicating a similar rate of phloem exudation (Fig. 3D). Furthermore, they had equal amino acid concentrations in the source leaf tissue (Fig. 3A), the principal starvation and exportable N pool that mainly originates from protein degradation and nitrate reduction (Dalling, 1985), and there was no difference between lines in leaf soluble protein concentration and NR activity.
Exudation in EDTA is currently accepted as a valuable and reliable technique for studying phloem sap composition (Lohaus et al., 1994), and compares satisfactorily with the aphid-stylet technique (Girousse et al., 1991; Chino et al., 1991; Weibull et al., 1990). In wheat plants the amino acid concentration in the phloem sap increases with increasing supply, and is dependent on the amino acid concentration in the leaf export pool (Caputo and Barneix, 1997). However, in these experiments the ditelosomic line failed to increase amino acid export in the phloem with increasing N supplies regardless of the N source (nitrate or ammonium). Similarly, under N deficiency the amino acid concentration in the phloem exudate decreased faster in CSDT7BL, although there were no differences between genotypes in leaf amino acid concentration until day two of N starvation (Fig. 5C, D).
Proteolysis is induced in dark-grown plants, increasing leaf amino acid concentration and export to the phloem (Caputo and Barneix, 1999). Thus, as expected, leaf amino acid concentration increased in both lines after several hours in the dark, but, only in the CS line did the amino acid concentration of the phloem exudate increase in response to dark treatment. This is consistent with the results of other experiments, where the only differences between genotypes, besides growth rate, is their export rate of amino acid in the phloem. The fact that no difference in sugar exudation rate was observed between the two lines indicates that the differences in amino acid exudation are not artefacts due to differences in leaves size.
These results indicate that the ditelosomic line has a deficiency in the export of amino acids in the phloem at high N supply, because the export of total amino acid reached a plateau at a lower external N concentration in CSDT7BL than in CS. This difference does not seem to depend on the form of nitrogenous supply, and it was observed in every experiment (Figs 3B, 4B, 5D, 6B). These results help to explain those previously reported for field conditions, where this line is inefficient in remobilizing N from the leaf to the grain under high fertilizer conditions (Fatta et al., 2000). In Brassica napus it has also been shown that phloem translocation of amino-N and the phloem loading of amino acids are decisive factors for the protein content in the seeds (Lohaus and Moellers, 2000).
The most abundant amino acids in the phloem of wheat plants are Glu, Asp, Gln, Ser, and Ala (Caputo and Barneix, 1997; Peeters and van Laere, 1994). The present results show that although no difference in the total amino acid composition of the leaf tissue of both lines were observed (Table 1), the phloem exudate concentration was different for some amino acids (Glu, Asp, Gln, Tyr, Thr, Ile Val, Ala, and Phe) while others did not change (Table 1). Hence in the ditelosomic line phloem loading of only some amino acids are discriminated against.
With the present results it cannot be determined at which point this discrimination is exerted. Further studies on the mechanism of amino acid loading into the sieve tubes are required. There are indications that a mechanism of transport against a concentration gradient, probably driven by a proton symport may be involved in the uptake of amino acids into the sieve tubes (Frommer et al., 1994). Genes encoding amino acid permeases expressed in mature plant leaves, may play a role in phloem loading (Rentsch and Frommer, 1996). At present, several families of amino acid transporters with different specificity are recognized (Fischer et al., 1998), which give a focus for further research into CSDT7BL deficiency.
The present results suggest that the CSDT7BL line has an impairment of the amino acids export in the phloem, probably in phloem loading. This impairment caused by the deletion of the short arm of chromosome 7B suggests that this chromosome arm carries genetic information important for the phloem loading of amino acids. The grain protein content in cereals depends mainly on amino acid remobilization from the vegetative organs to the ear via the phloem (Dalling, 1985), and although there are probably many important genes missing in the ditelosomic line, the present results suggest that the decrease in grain protein concentration observed in mature plants of CSDT7BL could be a consequence of the deficiency in amino acid export observed in this line. This ditelosomic line is shown to be a valuable tool for the study of the mechanism for amino acid loading to the phloem, and the physiology and genetics of the regulation of grain protein concentration in wheat.
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Acknowledgments |
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This research was supported by a grant from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) of Argentina.
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Notes |
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1 To whom corespondence should be addressed. Fax: +54 011 4514 8741. E-mail: barneix{at}mail.agro.uba.ar
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References |
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