JXB Advance Access originally published online on August 28, 2007
Journal of Experimental Botany 2007 58(12):3183-3195; doi:10.1093/jxb/erm164
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Uptake and allocation of carbon and nitrogen in Vicia narbonensis plants with increased seed sink strength achieved by seed-specific expression of an amino acid permease
1Fachgebiet Pflanzenbau in den Tropen und Subtropen, Humboldt Universität zu Berlin, D-14195 Berlin, Germany
2Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben, Germany
3Biology Department, Trent University, Peterborough, ON, K9J 7B8 Canada
4Universität Rostock, Medizinische Fakultät, Kinder- und Jugendklinik, D-18057 Rostock, Germany
* To whom correspondence should be addressed. E-mail: weber{at}ipk-gatersleben.de
Received 28 March 2007; Revised 13 June 2007 Accepted 26 June 2007
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Abstract |
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Over-expressing an amino acid permease in Vicia narbonensis seeds increases sink strength for N that is evident from the higher seed protein content and seed weight. Here, the effect of increased seed sink strength of line AAP-12 on growth, development, and on whole plant carbon and nitrogen uptake and partitioning is analysed. AAP-12 plants have a prolonged growth period. Accumulation and partitioning of dry matter and N in leaves, stems, and pods are higher whereas remobilization to the seeds is delayed, indicating that the switch from growth to reserve allocation and remobilization is delayed. Measuring uptake and allocation of 15N-ammonia applied via the roots revealed a higher and longer label uptake period during maturation. Measuring whole plant carbon fixation and allocation after 13C labelling shows higher levels at maturation, particularly in seeds, indicating higher seed sink strength for C and increased allocation into maturing seeds. Levels of cytokinins were dramatically increased in AAP-12 seeds indicating its role in nitrogen-mediated growth stimulation. AAP-12 seeds have higher natural abundances for 13C indicating increased C fixation via PEP carboxylase in order to meet the higher demand of carbon acceptors for amino acid synthesis. In summary, increased seed sink strength for N in AAP-12 stimulates seed growth, but also that of vegetative organs, which finally leads to a higher ratio of vegetative to seed biomass at maturity and thus a lower harvest index. Therefore, the increased N uptake due to higher seed demand of AAP-12 is partly compensated by growth stimulation of vegetative organs.
Key words: Amino acid permease, C and N partitioning, cytokinin, harvest index, legume seed development, seed sink strength, transgenic plants
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConcluding remarksReferences |
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Legume seeds are a major source of plant-derived proteins and are economically important for worldwide feed and food. Storage protein accumulation in legumes occurs in the embryo during maturation where nutrients like sugars and nitrogen confer regulatory control on storage activities (Weber et al., 2005). The nutrient status functions as part of a signalling framework which also involves the hormone abscisic acid and which stimulates storage protein production at the transcriptional level (Weber et al., 2005; Radchuk et al., 2006). Accordingly, storage protein accumulation in pea depends on nitrogen availability in the seed (Lhuillier-Soundélé et al., 1999; Golombek et al., 2001; Miranda et al., 2001; Salon et al., 2001). Also in maize and barley, endosperm-specific synthesis of storage proteins is under nutritional control and dependent on nitrogen availability (Balconi et al., 1991; Müller and Knudsen, 1993).
However, the increase in seed protein content seems to be source and sink regulated, for example, it can result either from an improved capacity of the grain to accumulate N or from a greater N supply to the seeds by uptake from the soil and/or remobilization from vegetative organs (Gastal and Lemaire, 2002; Martre et al., 2003; Barneix, 2007). There is evidence that seed N accumulation is more regulated by the supply of N rather than by seed activity (Triboi and Triboi-Blondel, 2002; Martre et al., 2003).
It has been reported that the level of sink demand for carbon and nitrogen can feed back through uptake, translocation, and assimilation rates (Peoples and Gifford, 1990). Enhancing sink strength can increase utilization of photosynthesis products and may enhance photosynthetic output and growth (Paul and Foyer, 2001). In Arabidopsis the nitrogen status of the whole plant controls uptake of N via long-distance signalling (Lejay et al., 1999). Maize cultivars with a higher seed protein content assimilate more nitrogen in the leaves (Lohaus et al., 1998;Lohaus and Moellers, 2000). Thus, seed sink strength can also stimulate source activity. The N status of the plant can be communicated by cytokinins (CKs) (Simpson et al., 1982; Collier et al., 2003; Sakakibara et al., 2006) via a molecular mechanism on N-dependent CK biosynthesis (Miyawaki et al., 2004; Sakakibara, 2006). In some cases this leads to a clear stimulation of growth (Rahayu et al., 2005). However, these studies mainly addressed the situation in shoots and roots whereas much less is known for seeds.
The amount of N taken up by the crop has a major impact on overall crop growth rate (Gastal and Lemaire, 2002). Leaf photosynthesis increases together with leaf N content and a significant effect of leaf N content on light use efficiency has been reported (Grindlay, 1997). It has been concluded that the impact of N supply on leaf growth was mostly due to increased cell growth rate with larger cell size (Gastal and Lemaire, 2002). Plants accumulate N mainly during the vegetative period of growth and after anthesis N uptake rates rapidly slow down. Moreover, soybean and common bean followed distinct patterns of N accumulation over time. About 50% of the total N accumulation by common bean occurred during vegetative growth, a period which accounted for 56% of its total growth duration, indicating that growth duration and N accumulation were nearly proportional. By contrast, only 28% of the total N accumulation of a soybean cultivar was assimilated during vegetative growth, a period that accounted for 44% of the total growth period. The early reproductive period was the period of most rapid N accumulation when 57% of the total N was assimilated in 25% of the growth duration (George and Singleton, 1992). Thus, the majority of N translocated to the seeds, up to 70%, is remobilized from the vegetative pools (Salon et al., 2001; Schiltz et al., 2005).
The current knowledge shows that N uptake and utilization in crops are highly interrelated and involve many aspects of growth and development. Thus it is necessary to study uptake, assimilation, and distribution of nitrogen in the vegetative and the reproductive parts of crop plants as the key processes involved in the seed protein performance. To improve grain production in terms of yield, plants have to take up and accumulate more N and should also be able to use this N for grain growth and storage. Without an increase in grain N accumulation, the higher N uptake would result in a lower harvest index (Sinclair, 1998). For future crop improvement it is important to reach a deeper understanding of the relationship between uptake and delivery of assimilates to seeds, and, especially, of the role of vegetative organs and their transiently stored reserves related to seed filling.
In legumes, seed protein accumulation can be controlled by the capacity of the seed to import amino acids via specific transporters. In V. faba seeds, sink strength for nitrogen is acquired during maturation and is associated with amino acid transport (Golombek et al., 2001). Over-expressing an amino acid permease (VfAAP1; Miranda et al., 2001) in Vicia narbonensis seeds increases seed sink strength for nitrogen and leads to higher seed protein concentration and seed weight brought about by a higher capacity for amino acid uptake (Rolletschek et al., 2005). However, how far this increased capacity can be realized, for example, translated into higher seed protein, depends on other factors such as N uptake from the soil, N fixation and allocation, as well as remobilization from vegetative tissues. In this work, the amino acid permease over-expressing line AAP-12 was analysed and the effect of its increased seed sink strength on growth and development as well as on carbon and nitrogen uptake and partitioning was assessed. It was found that increased seed sink strength for N stimulates vegetative and seed growth which finally leads to a higher ratio of vegetative to seed biomass at maturity and thus to a lower harvest index. As a result, increased N uptake due to higher seed demand of AAP-12 is partly compensated by growth stimulation of vegetative organs.
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Materials and methods |
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Plant material
Vicia narbonensis, transgenic line AAP-12 (Rolletschek et al., 2005) and wild-type control plants, were grown in 3.0 l pots in growth chambers with 16/8 h light/dark at 20/18 °C. Plants were fertilized once a week with 50 ml of a 0.3% complete nutrient solution (Hakaphos®, blau, COMPO, Münster, Germany) with nitrate and ammonium in order to keep non-limiting N conditions. For the experiment described in Fig. 2, plants were grown to maturity in a substrate bed under glass from March to June 2005 without any additional light or temperature regulation in order to create semi-controlled conditions. The substrate was a mixture of humus and fine sand. Plants were not fertilized during the experiment but irrigated every two days to maintain an optimal water supply. Under normal conditions symbiotic N fixation is very important for grain legumes. However, for simplicity reasons, symbiotic N fixation was not considered for the analysis of the AAP-12 plants. The substrate and seeds were not inoculated with rhizobia. Accordingly, the control of the roots only revealed a very sporadic occurrence of nodules.
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In vivo labelling with 13C-CO2
All labelling was done in greenhouses with sun shelter installed to avoid temperatures above 25 °C; and additional light was given when necessary. Unlabelled control plants were used. Due to the 15N/13C labelling and the high enrichments above natural abundances of the plant tissues, it was concluded that discrimination processes play no role with response to the results. The appearance of the first flower was set as day one (1 DAP), after which an exact counting was made and given as DAP.
Wild-type and AAP-12 plants at different stages of development were covered with transparent plastic bags (40x150 cm). The plants were pulse labelled with 13C barium carbonate (99 atom% 13C) for 60 min. 13C-CO2 was generated by injecting 5 ml of 2 M HClO4 into a beaker with 1 g Ba13C-CO3. Plastic bags were then removed and plants cultivated for further 4 d following extraction and measurement of 13C in the different organs. All 13C measurements were performed using the mass spectrometer Tracer mass 20-20; SerCon, Crewe, UK, and total 13C as well as the 13C concentration was calculated.
Labelling of plants with 15N ammonia
The experiments described in Figs 3–8
were carried out in the greenhouse under long-day conditions (16/8 h regime). Plants were grown in 3.0 l pots in substrate with non-limiting N supply. Leaves, stems, pods, and seeds were analysed at flowering time (only leaves and stems) as well as at 20, 25, 33, and 40 d after pollination (DAP). At each stage of development plants were pulse labelled with 3 (or 6) mmol of 15N-NH4 in 200 ml water applied via the root system to at least five plants, either wild-type or AAP-12. Ammonia was applied as the tracer. Moreover, ammonia was used to avoid possible limitations due to differences in the reduction capacity (Barneix, 2007) of AAP-12 and wild-type plants.
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Four days later, the plants were harvested and analysed for total dry matter, percentage of nitrogen content, total N content, 15N concentration, and total 15N content. The 15N uptake rates for the different organs (mg 15N excess d–1) were calculated using the values in Fig. 7M–P.
N and C measurements were carried out as described in Weber et al. (2000) and Götz and Herzog (2000). Compared with experiment 2, where the plants were grown in the greenhouse in early summer at a higher temperature, experiment 3 was carried out in the early spring at a lower temperature.
13C and 15N signatures
Aliquots of the dry plant material (1–2 mg) were weighed into tin capsules and analysed for total N, C, and atom% C and N. Measurements were performed by KD Wutzke, Research Laboratory, Children's Hospital, University of Rostock, Germany using the mass spectrometer Tracer mass 20-20; SerCon, Crewe, UK.
Cytokinin determination
CKs were extracted, purified, and quantified by an isotope dilution assay under the conditions described by Ferguson et al. (2005). Deuterated CKs, [2H6]iP, [2H6][9R]iP, trans-[2H5]Z, [2H3]DZ, trans-[2H5][9R]Z, [2H3][9R]DHZ, [2H6][9R-MP]iP, and [2H6][9R-MP]DHZ (OlChemIm Ltd, Olomouc, Czech Republic) were added as quantitative internal standards. Nucleotides were converted to nucleosides for quantification. Purified CK were separated and analysed by (LC-(+)ESI-MS/MS) using a Waters 2680 Alliance HPLC system (Waters, Milford, USA) linked to a Quattro-LC triple quadrupole MS (Micromass, Altrincham, UK).
Nucleic acid isolation and hybridization techniques
Nucleic acids were isolated and northern and Southern hybridizations were performed as described in Heim et al. (1993). The VfAAP1 cDNA fragment was used as probe after labelling with 32P-dCTP as described in Miranda et al. (2001).
Statistical treatment
If not indicated otherwise, the data were given in means±standard error (SE) or ±standard deviation (SD). Significance levels were calculated according to Student's t test, (*), P <0.05.
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Results |
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Molecular characterization of line AAP-12
Southern gel blot analysis was performed in order to determine the transgene copy number in the line AAP-12. DNA extracted from seven transgenic and four wild-type plants was restricted, gel-separated, and hybridized with a VfAAP1 cDNA-probe. From all transgenic plants, a single hybridizing band is evident (Fig. 1A, lanes 1–7, asterisk), indicating a single copy insert. Two other bands appear for both the AAP-12 (lanes 1–7) and the wild-type plants (lanes 9–11) and may represent cross-hybridizing endogenous amino acid permeases.
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The expression of VfAAP1 in the embryo was checked by northern gel blot analysis using embryos of AAP-12 at 21, 24, 30, and 35 DAP. VfAAP1-mRNA levels increases from 21 DAP to 30 DAP reflecting the activity profile of the LeB4 promoter (Fig. 1B). In the wild-type control only a faint band is visible which probably corresponds to the gene expression of the V. narbonensis endogenous amino acid transporter (Fig. 1B). A related quantification of the VfAAP1 expression profile is shown in Fig. 1C.
Yield-related parameters of line AAP-12
In experiment 1, yield related parameters were determined using a set of 10 mature plants of line AAP-12. Ten wild-type and ten AAP-12 plants were grown to maturity in a substrate bed in a greenhouse from March to June 2005, without additional light or temperature regulation in order to create semi-controlled conditions. The AAP-12 plants had a higher total biomass (Fig. 2A) and vegetative biomass (Fig. 2B). Pod number per plant (Fig. 2C), seed number per pod (Fig. 2D), seed number per plant (Fig. 2E), and seed weight per plants (Fig. 2F) were not significantly different although values of seeds per plant and seeds per pod showed a trend to lower levels. From the values, the ‘harvest index’ (ratio of grain dry matter to total dry matter) was calculated, which was lower for AAP-12 plants, 0.34 compared with 0.4 for the wild type (Fig. 2G). As shown before with phytochamber-grown plants, individual seed weight (Fig. 2H) and percentage of seed nitrogen (Fig. 2I) were approximately 10–15% higher, resulting in a higher seed nitrogen content per plant (Fig. 2J). Seed carbon was not changed (Fig. 2K) resulting in an increase of the seeds N to C ratio (Fig. 2L).
Seed development
Developmental parameters were compared for AAP-12 and wild-type seeds in terms of fresh weight (Fig. 3A) and dry weight accumulation (Fig. 3B). Both dry and fresh weight accumulation display a similar pattern over time. Levels are not different at 20 DAP, but are higher at 25–40 DAP, which represents the maturation phase. This indicates increased seed filling rates of AAP-12 seeds. The water content of the embryo represents a measure for the developmental state. As dry weight accumulation proceeds there is a parallel decrease of the relative water content (Weber et al., 2000). However, there were no significant differences in water content at 20–40 DAP between the AAP-12 and the wild-type embryos (Fig. 1C).
AAP-12 seeds have increased cytokinin levels
It was shown that AAP-12 seeds contain more total nitrogen, storage proteins, and have higher growth rates and individual seed weight (Rolletschek et al. 2005). Therefore, the levels of cytokinins in AAP-12 and wild-type seeds were analysed at six stages of development. Total CK levels are highest in 15 DAF seeds, which then rapidly decreased by around 70% after 17 DAP. Interestingly, in AAP-12 seeds CK levels were dramatically higher, around 10-fold, at all stages analysed (Fig. 4). The predominant CKs forms that are increased include several forms that are normally considered active: [9R]DHZ, trans-[9R]Z, [9R]iP and their corresponding nucleotides. This indicates the involvement of CKs in the N-mediated growth stimulation of AAP-12 seeds.
Dry matter accumulation and partitioning on a whole plant level at 33 DAP and 40 DAP
In experiment 2 (labelling periods in the greenhouse) dry weight as well as total nitrogen distributions, as determined by the Kjeldahl method, have been determined at 33 DAP and 40 DAP (first flower) in leaves, stems, pods, and seeds. At 33 DAP, the AAP-12 plants contained 12% more total dry weight than the wild type (Figs 5A, 6A). However, the wild-type plants invested a significant higher portion of dry matter into the seeds, 42% versus 31% (Fig. 6A), whereas in the AAP-12 plants absolutely and relatively more dry matter was incorporated into the vegetative parts, particularly into stems, 30% versus 22% (Figs 5A, 6A).
At 40 DAP, dry matter content of the wild type was not much different from that at 33 DAP (Fig. 6A, B). However, the relative distribution revealed a shift in favour of the seeds, 42% versus 46% at 33 DAP and 40 DAP, respectively. For the wild type, this indicates stronger remobilization of vegetative dry matter to the seeds between 33 DAP and 40 DAP rather than net accumulation of dry matter. By contrast, from 33 DAP to 40 DAP the AAP-12 plants accumulated an additional 11% of dry matter (Figs 5A, B, 6A, B) indicating a prolonged growth and dry matter accumulation period. Dry matter was partitioned into leaves, stems, and seeds which were significantly higher compared with the wild type (Fig. 5B). The portion of dry matter partitioned to the seeds increased from 31% to 42% between 33 DAP and 40 DAP in the AAP-12 plants (Fig. 6A, B).
Nitrogen accumulation and partitioning on a whole plant level at 33 DAP and 40 DAP
Total nitrogen content in wild-type plants from 33 DAP and 40 DAP did not significantly change (Figs 5C, D, 6C, D) indicating that N uptake had already ceased. However, remobilization still took place from 33 DAP to 40 DAP, which is reflected in an increasing proportion of seed N, from 50% to 58% (Fig. 5C, D). At 33 DAP, the AAP-12 and wild-type plants contained the same amount of N (Fig. 6C). However, in the AAP-12 plants a higher proportion of that N was present in the vegetative organs, 54% versus 49% (Figs 5C, D, 6C, D). In the AAP-12 plants there was a further increase of total N by 14% from 33 DAP to 40 DAP (Fig. 6C, D) indicating a prolonged uptake period of available N. At the same time remobilization leads to an increase of the seed N proportion from 46% to 58% (Fig. 6D). Interestingly, at 40 DAP the total N content of the AAP-12 was 26% higher than the wild type, but there was no large alteration in the relative proportion between organs. This indicates that in the AAP-12 at 33–40 DAP nitrogen uptake is higher rather than allocation.
Uptake and allocation of 15N-ammonia applied to roots
A pulse label of 3 mmol of 15N-NH4 was applied to the roots of AAP-12 and wild-type plants at 29 DAP and 36 DAP. Four days later the plants were harvested and the 15N label was measured in the different organs. At 33 DAP the AAP-12 took up 67% more label compared with the wild type and 70% of that label was present in the seeds, compared with 64% in the wild-type seeds (Figs 5E, 6E). In another experiment using 6 mmol 15N-NH4, the label uptake was even 100% higher for the AAP-12 plants (data not shown). At 40 DAP the AAP-12 plants even took up 122% more label than the wild type (Figs 5F, 6F). That means that label uptake activity into wild-type plants decreased more rapidly between 33 DAP and 40 DAP than for the AAP-12 plants. Interestingly at 40 DAP, 79% of the label taken up into the AAP-12 plant was found in the seeds (Fig. 6F), whereas in the wild type only 20% of total label was present in seeds and most stayed in the stem, 57% (Figs 5F, 6F). That means that, at 40 DAP, the wild-type plants can still take up 15N-ammonia via the roots, although at lower rates than the AAP-12 plants, but wild-type plants are not able to translocate the 15N to the seeds efficiently. On the other hand, AAP-12 plants at 40 DAP have both a higher 15N-NH4 uptake rate and a higher translocation activity into seeds.
The experiment was repeated using a pulse of 6 mmol 15N-NH4 applied to the roots with essentially similar results (data not shown).
Dry matter and nitrogen partitioning between 0 DAP and 40 DAP
Sampling in experiment 3 (labelling periods in the greenhouse) was done at flowering, 20, 25, 33, and 40 DAP. Dry weight levels for leaves did not change over time, with only slightly higher levels for the AAP-12 plants at 25 DAP (Fig. 6A). This was different for stems where, except for the flowering stage, levels were significantly higher (up to 30%) in the AAP-12 plants from 20–40 DAP (Fig. 6B). A similar trend was observed for the pods; that are higher values for AAP-12 plants from 25–40 DAP (Fig. 6C). Dry matter in seeds were similar in both plant types at 20 DAP and 25 DAP, but were higher in AAP-12 plants at 33 DAP and 40 DAP (Fig. 6D).
Percentages of nitrogen were not largely different for AAP-12 leaves although there was a trend to slightly lower levels for the AAP-12 plants. At 25 DAP levels were even significantly lower (Fig. 7E). In AAP-12 stems N concentration was lower from 25 DAP onwards (Fig. 7F). The percentages of nitrogen were not different in AAP-12 pods at 20 DAP and 25 DAP, but were lower at the later stages (Fig. 7G). For the seeds there was no significant change at all the stages analysed (Fig. 7H). Calculating the total nitrogen content revealed non-significant difference for leaves, stems, and pods although a significant higher level occurred in AAP-12 stems at 20 DAP, which was non-significant at other stages (Fig. 7I, J, K). Total nitrogen in seeds was not different at 20 DAP and 25 DAP but significantly higher in the AAP-12 plants at 33 DAP and 40 DAP (Fig. 7L).
Plants were pulse labelled with 6 mmol 15N-ammonia via the roots. Four days later the 15N label was measured in the different organs. In leaves, stems, and pods the total amounts of 15N were not different, with the exception of lower levels in the AAP-12 stems at 25 DAP. However, for the AAP-12 stems and pods there was a general trend to lower levels, but due to large standard deviations these differences were mostly not significant (Fig. 7M, N, O). For the seeds there was a higher content of 15N in AAP-12 at 33 DAP which represents the main storage phase (Fig. 7P). From the data of Fig. 7M–P, the 15N uptake rates were calculated. For leaves, stems, and pods, levels were highest at youngest stages with an opposite trend in seeds. 15N uptake rates in AAP-12 seeds were higher around 33 DAP, the main maturation phase. At the same stage levels are lower in AAP-12 stems (Fig. 8).
In summary, the results from experiment 3 demonstrate that AAP-12 plants realized higher seed nitrogen levels. However, this was largely due to an increased dry matter accumulation, which means that the nitrogen concentration is not different. Furthermore, the results confirmed our earlier finding that increasing the seed sink strength for nitrogen stimulates vegetative as well as seed growth which leads to higher dry weight levels in both organs.
In vivo labelling with 13C-CO2
In order to assess carbon fixation and allocation, plants were labelled with 13C-CO2 at 16 DAP and 29 DAP following cultivation for another 4 d and measurement of 13C. At 20 DAP, there was no difference in the total uptake of 13C between AAP-12 and wild-type plants, 8.48 and 8.35 mg 13C g–1 dry weight, whereas, at 33 DAP, the AAP-12 plants took up more 13C, 9.52 versus 8.51 13C g–1 dry weight. In leaves, 13C levels were not different at 20 DAP and were higher in the wild type at 33 DAP (Fig. 9A). In stems, levels were not different at 20 DAP and 33 DAP (Fig. 9B). The 13C levels in pods were higher at 33 DAP but unchanged at 20 DAP (Fig. 9C). Seeds accumulated equal amounts of 13C label at 20 DAP, but levels were higher in AAP-12 seeds at 33 DAP (Fig. 9D). These results indicate an increased allocation of labelled carbon into maturing AAP-12 seeds.
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13C and 15N signatures
Variations in the distribution of stable isotopes of C and N can give evidence for acquisition strategies and isotopic discrimination of key enzymes resulting in end-products with different isotopic composition (Gleixner et al., 1998). The natural abundance of 13C and 15N in AAP-12 and wild type was measured in a fraction of dry mature seeds. As shown before, AAP-12 seeds contained more nitrogen (Fig. 2I) on a dry matter basis, whereas the carbon content was unchanged (Fig. 2K). The
15N signature was not different (Fig. 10A). However, the 13C value was less negative for the AAP-12 seeds (Fig. 10B) indicating that these seeds contain more 13C and indicating that carbon in AAP-12 seeds may partly originate from higher carbon fixation via PEP carboxylase in seeds.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConcluding remarksReferences |
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Legume seeds are a major source of plant-derived proteins. However, in practice, seed protein content and yield are often variable and insufficient (Salon et al., 2001). For improvement it is necessary to increase uptake and accumulation of N from the soil and allocation and remobilization of that reserve to the seeds (Sinclair, 1998; Triboi and Triboi-Blondel, 2002). In legumes, seed protein accumulation is a function of available N to the seed (Lhuillier-Soundélé et al., 1999; Salon et al., 2001). Therefore, increasing the ability of the seed to attract N may be crucial. V. narbonensis plants were recently created which over-express an amino acid permease from V. faba in the seeds (VfAAP1; Miranda et al., 2001) and it could be shown that transgenic seeds have increased sink strength for N, and higher seed protein concentrations and seed weight (Rolletschek et al., 2005). Because it is known that higher sink demand interacts with assimilate uptake and allocation as well as overall plant growth and development, the VfAAP1 over-expressing line AAP-12 was analysed here and the effect of increased seed sink strength on plant growth and the distribution of C and N on the whole plant level was assessed.
AAP-12 over-expression stimulates growth and decreases harvest index
Experiment 1 was performed with plants grown under semi-controlled conditions. Mature AAP-12 seeds have increased seed N concentration (Fig. 2I), seed N per plant (Fig. 2J), and single seed weight. However, total biomass and vegetative biomass of AAP-12 plants are significantly higher (Fig. 2A, B). This finally leads to a decrease of ‘harvest index’, expressed as grain dry matter per total plant dry matter. It is speculated that the higher seed sink strength of AAP-12 caused stimulation of growth responses at the whole plant level including the vegetative organs, which both have higher fresh and dry weight accumulation rates (Fig. 2A, B).
In experiment 2 (greenhouse), dry matter accumulation and partitioning in different organs was measured at 33 DAP (mid maturation) and at 40 DAP (end of maturation). Again, AAP-12 plants accumulate relatively and absolutely more dry matter whereas wild-type plants had already stopped growth at 33 DAP and during the period measured distribute relatively more dry matter to the seeds (Figs 4A, B, 5A, B). It is concluded that between 33 DAP and 40 DAP the AAP-12 plants continue to grow whereas, in the wild type, dry matter does not increase but is allocated to the seeds. Thus, AAP-12 plants have a prolonged growth period.
N distribution shows a similar pattern. Because the AAP-12 plants accumulate N for a longer period this leads to a further increase of N between 33 DAP and 40 DAP. In the wild type, N uptake had already ceased at 33 DAP and N allocation to the seeds takes place from 33 DAP to 40 DAP. In summary, the result from experiment 2 shows that, in AAP-12 plants, the shift from growth to reserve allocation and remobilization is delayed. The results indicate that there is a particular dynamic of N accumulation and mobilization and plant growth that is caused by changing patterns of source–sink interactions (Atta et al., 2004).
Measuring uptake and allocation of 15N ammonia at 33 DAP and 40 DAP reveals an absolutely higher uptake into the AAP-12 plants (Figs 5E, F, 6E, F). In addition, the 15N uptake capacity in AAP-12 decreased more slowly and plants were able to translocate the 15N more efficiently to the seeds, at both 33 DAP and 40 DAP. By contrast, the wild type drastically lost the capacity to allocate 15N to the seeds between 33 DAP and 40 DAP, possibly because seeds reached maturation earlier and the phloem connections were degrading. At 40 DAP, wild-type plants allocate only 20% of total 15N label taken up to the seeds, in contrast to AAP-12, where nearly 80% is allocated. The prolonged capacity for N uptake and allocation to seeds of AAP-12 is in accordance with an increased activity of amino acid transport (Rolletschek et al., 2005).
In experiment 3 (greenhouse), dry matter and N distribution were measured at five stages starting at flowering. Compared with experiment 2, where the plants were grown in the greenhouse in the early summer at higher temperatures, experiment 3 was carried out in the early spring at lower temperatures, which slowed down seed development; a discrepancy which prevents direct comparisons between experiments 2 and 3. In general, differences are only evident at seed maturation and not at the flowering stage, indicating a specific effect of the transgene. This is due to the activity profile of the LeB4 promoter, which drives transgene expression and which is active during early to late seed maturation. Again, a similar stimulation of growth was observed for AAP-12, especially for stems, pods, and seeds (Fig. 7A–D). In general, N content in all vegetative organs decreased during seed development. For AAP-12 and the wild type, N levels are not different in leaves, stems, and pods (Fig. 7I, J, K), whereas the N concentration is lower in AAP-12 stems, pods, and leaves (only at 25 DAP) but unchanged in seeds (Fig. 7E–H). Due to the higher dry matter accumulation of the AAP-12 (Fig. 7D), seeds contain significantly more N at 33 to 40 DAP (Fig. 7L).
In summary, from experiment 3 it can be concluded that (i) the lower N concentration in stems and pods from 20 DAP onwards may be caused by an increased N demand of the seeds due to over-expression of VfAAP1, (ii) a close interaction exists between N uptake and allocation and growth processes in both vegetative and seed organs, and, (iii) higher seed N demand increased N content rather than N concentration because stimulated growth leads to a compensatory effect. For pea it has also been shown that feeding with high NO3 increased plant dry weight (Atta et al., 2004), indicating a general effect of nitrogen on growth in legumes.
Labelling of plants with 15N ammonia always revealed the highest contents in young vegetative organs at the flowering stage (Fig. 7M–P). Similar results have been obtained for V. faba and pea (Dekhuijzen and Verkerke, 1984; Atta et al., 2004; Schiltz et al., 2005). During seed maturation there was no major difference between AAP-12 and wild-type organs except for lower levels in AAP-12 stems at 25 DAP (Fig. 7N). However, AAP-12 seed levels were higher at 33 DAP (Fig. 7P). The 15N uptake rates (Fig. 8) revealed that, during seed maturation, leaves, stems, and pods generally lose their ability for 15N uptake whereas that of the seeds increased. Higher 15N uptake rates of AAP-12 seeds and concomitant lower rates of stems at mid maturation (Fig. 8) are again in accordance with higher seed sink strength for N.
It has been shown that increased seed N demand can generate long-distance signals within the plant, which stimulate N uptake in the roots via N-transporters (Gansel et al., 2001). Studies with Arabidopsis (Lejay et al., 1999) have shown that the nitrogen status of the whole plant can control uptake of mineral N via long-distance signalling (Tillard et al., 1998). Whether such mechanisms act in the AAP-12 plants is currently not known and awaits further investigations. Possibly certain N compounds or amino acids could be translocated through the phloem and may signal the nitrogen status of the plant. An increased N uptake can be associated with the stimulation of growth processes, which possibly involves cytokinins (Rahayu et al., 2005; Sakakibara et al., 2006). Therefore, increasing nitrogen uptake or elevating the N status can be coupled to growth.
It is clear that, under natural conditions, legume plants mainly rely on symbiotic nitrogen fixation. From the data presented here it is difficult to predict what would happen under such conditions. Eventually, increased seed sink strength for N might stimulate N fixation. However the high energy costs under such a situation might lead to carbon/energy limitation. Future experiments under more natural conditions should help to resolve this question.
It has been shown that total cytokinin levels are dramatically increased in AAP-12 seeds. This strongly indicates a role for CKs in the N-dependent growth stimulation of AAP-12 seeds. Similarly, the analysis of pea embryos also reveals a strong growth-promoting effect for CKs (Quesnelle and Emery, 2007). In particular, for roots, it has been shown that CKs can communicate the N demand of the shoot and that CK synthesis is a response to increased nitrogen (Simpson et al., 1982; Takei et al., 2001, 2002). Similarly, atmospheric N deposition results in the activation of cytokinins in the shoot and increased shoot-to-root transport of cytokinins in the phloem (Collier et al., 2003).
In the AAP-12 seeds a similar situation can occur. Increased N uptake due to AAP1 overexpression may lead to higher CK levels, and this can stimulate growth processes. Whether the higher CKs concentrations in the AAP-12 seeds are derived from the vegetative tissue or are directly synthesized in the seeds is currently unknown. However, in Arabidopsis, genes for CK biosynthesis are expressed in the endosperm (Miyawaki et al., 2004). Moreover, CK budgets of legumes indicate seeds are largely self-sufficient for CK and they may even act as CK sources for vegetative organs (Emery and Atkins, 2006). Therefore, the AAP-12 activity increased seed N, which, in turn, probably stimulated a large CK biosynthetic increase in situ.
AAP-12 over-expression stimulates also C allocation to seeds
Measuring whole plant C fixation and allocation using 13C-CO2 feeding at 20 DAP and 33 DAP shows that at early seed development (20 DAP) total 13C uptake and distribution is not different (Fig. 9). However, at 33 DAP the AAP-12 plants have accumulated 12% more 13C. This is mainly due to the seeds, which alone contain 50% more 13C than wild-type seeds, whereas the 13C content in the AAP-12 leaves and stems are even lower, possibly due to an increased translocation to the seeds. In summary, it is concluded that the higher seed sink strength for N also caused a higher seed uptake of carbon either as carbohydrates or as amino acid backbones.
Variations in the distribution of stable isotopes of C and N give evidence for isotopic discrimination of key enzymes resulting in end-products with different isotopic composition (Gleixner et al., 1998). In mature AAP-12 seeds the 13C value was less negative (Fig. 10B) indicating that these seeds have a higher natural abundance for 13C. Seed carbon mainly comes from the CO2-fixing enzymes Rubisco and/or PEP carboxylase. In contrast to Rubisco, PEP carboxylase does not discriminate between 13CO2 and 12CO2. Thus, a less negative 13C value occurs when a higher percentage of carbon is fixed by PEP carboxylase (Melzer and O'Leary, 1987). A possible explanation is that, for AAP-12, the higher seed N import, especially of amides, requires carbon acceptors, which may be supplied by PEP carboxylase, which will cause a higher C flux through this enzyme (Golombek et al., 1999). This is also in accordance with the increased activity of PEP carboxylase in lines over-expressing VfAAP1 in the seeds (Rolletschek et al., 2005). Differences of 15N values can reflect different nitrogen acquisition strategies, metabolism, and losses (Wanek and Arndt, 2002). However, the natural abundance of 15N is not different between AAP-12 and wild-type seeds.
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Concluding remarks |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConcluding remarksReferences |
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These results show that, in the V. narbonensis line AAP-12, increased seed sink strength for N stimulates vegetative growth which leads to a higher ratio of vegetative to seed biomass at maturity and thus a lower harvest index. It is concluded that increased N uptake due to higher seed demand of AAP-12 is partly compensated by growth stimulation of the vegetative organs. Stimulation of vegetative growth and prolonged seed development covered the increased N-accumulation so N% remained unchanged.
Obviously the primary reason for the changes is the overexpression of the amino acid transporter in the seed. It is difficult to predict the relevance of amino acid permease overexpression in terms of agricultural productivity. It could be shown that the higher N uptake into the plants stimulated growth both of vegetative and seed tissues. Therefore it cannot be concluded that the improved nitrogen content is partly due to extended growth. On the other hand, percentage of seed N is increased (Fig. 2I) which points to a direct effect of the amino acid permease. Recent work on yield physiology of soybean revealed that, in general, seed fill duration is correlated to yield (Egli, 1994). Hanson and Burton (1994) selected soybean genotypes for increased seed fill duration and identified a complex genetic system associated with delayed or extended seed maturation which was associated with a reduction of both specific dry matter accumulation and specific seed density. A desirable genotype should have a rapid early seed fill to establish seed set and a reduced seed maturation rate occurring later in development utilizing favourable seed-filling conditions (Hanson, 1992).
Other research has also shown that there is often a great deal of compensation among seed mass, seed number, and seed growth rate as well as compensation by altered seed fill duration and overall plant growth. Finally, there is no benefit in yield or harvest index (Sinclair, 1998). Therefore, care has to be taken when transgenic approaches are applied with the aim of improving agronomic values of crop plants.
Sinclair (1998), recommends common features essential in achieving success: early assessment of the putatively beneficial trait, effective phenotyping of genetic modifications, multi-disciplinary effort and long-term commitment. Nevertheless, transgenic plants like the AAP-12 are valuable tools with which to study the physiological and molecular effects of altered seed sink strength or metabolism, growth, and development at the seed and the whole plant level.
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Acknowledgements |
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We are grateful to Katrin Blaschek, Elsa Fessel, Angela Schwarz, and Susanne Moryson for excellent technical assistance. We thank Isolde Saalbach for the help in plant transformation and Ulrich Wobus for discussions and continuous support. This work was supported by the European Union (Integrated project GRAIN LEGUMES) and Deutsche Forschungsgemeinschaft (DFG, Schwerpunktprogramm 1108, Transport Processes in Plants) and NSERC, Canada.
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