Co-occurrence of organic and inorganic N sources influences asparagine uptake and internal amino acid profiles in white clover

Direct plant uptake of organic nitrogen (N) is important for plant N nutrition, but we lack knowledge of how the concentration of external N forms (organic and inorganic) -influence organic N uptake and plant N status. We investigated the uptake of the amino acid asparagine (Asn) in white clover in the presence of different nitrate (NO3-), Asn, and total N concentrations. White clover seedlings were for one week exposed to combinations of NO3- (3-30 µmol N kg-1 sand DW) and Asn (3-30 µmol N kg-1 sand DW), where after the Asn uptake rate was determined by addition of 13C4-Asn. Shoot and root Asn content and amino acid profiles were also analyzed. Increasing external NO3- and total N concentrations decreased 13C4-Asn uptake rates and internal clover Asn content. Furthermore, total N affected clover amino acid profiles from non-essential amino acids at low N doses to the dominance of essential amino acids at increasing N doses. Asn uptake rate in white clover is reduced by increasing inorganic N, but not by increasing organic N concentrations. Furthermore, plant amino acid profiles are likely to be a more sensitive indicator of N supply and descriptor of the N status.


36
Legumes (Leguminosae) are of great significance to human, food, and animal feed due to their high 37 nitrogen content and are mainly grown as grain, and forage species (1). They are rich in amino acids that are assimilated 38 or derived from N accessed in three different processes: (1) N 2 -fixation, (2) inorganic N uptake (NO 3 -, NH 4 + ), (3) 39 organic N uptake (amino acids) (2, 3). N 2 -fixation and inorganic N uptake have been widely studied in legumes (4-6).

40
In terms of energy cost, amino acid assimilation through these two pathways is the most-energy demanding. While 41 direct uptake of organically bound N, where N is already in reduced form, costs less energy (7). Moreover, carbon cost 3 50 advance our understanding on how much amino acids contribute to the legume N budget, plant growth should be 51 compared in soils that differ in N quality and quantity.

52
Information on the co-occurrence of different N forms and their influence on root absorption of amino acids mainly 53 comes from studies with non-legume crop and tree species. One of the common findings is that uptake of amino acids is 54 increased, while the absorption of inorganic N is reduced in mixtures of different N sources. Perennial ryegrass (Lollium 55 perenne) exposed to a single and equimolar mixture of N sources (2 mM total N) doubled the uptake of glycine when 56 supplied with NO 3and NH 4 + compared to when supplied alone (12). Spring wheat (Triticum aestivum L. cv. Amaretto) 57 downregulated the assimilation of NO 3when exposed to a mixture of glycine (1 mM) and NO 3 -(1 mM), while the 58 acquisition of the amino acid was unaffected by the NO 3 -(13). Pre-incubation of young spruce (Picea abies L.) and 59 beech (Fagus sylvatica L.) with amino acids (10 mM) reduced the NO 3uptake when exposed to NO 3and NH 4 + (300-60 600 µM), whereas the root amino acid content increased (14). Similar results were obtained for non-mycorrhizal beech 61 (Fagus sylvatica L.), which was fed with NO 3 NH 4 with and without amino acids: uptake of organic N was significantly 62 higher than inorganic N (15). On the contrary, other studies on temperate and tropical forests point to a preferential 63 uptake of inorganic rather than organic N from mixtures different N sources (16,17). Whether such interactions occur 64 in legumes is unknown. However, a significance of amino acid uptake in the presence of inorganic N has been 65 documented for white clover (Trifolium repens). Based on the detection of L-asparagine-13 C 4 , 15 N 2 , we reported uptake 66 rates of 0.4 and 0.04 µmol g -1 root DW h -1 in a sterile hydroponic solution in both the presence and absence of NH 4 NO 3

67
(3). Clover plants were also shown to compete for amino acids under soil conditions with uptake rates between 0.05 and 68 0.15 µmol g -1 root DW h -1 (9). Hence, amino acids can constitute a significant portion of N that is acquired by legumes, 69 but we lack knowledge on the uptake of organic N in the presence of different N mixtures.

70
N uptake is thought to be strongly regulated by the N demand of the plant, specifically by the pool of amino compounds 71 circulating between shoot and roots (7). Correspondingly, changes in amino acid composition can be affected by the N 72 supply (18). This was observed by Cambui et al. (2011) who reported that in Arabidopsis thaliana grown on a mixture 73 with NO 3 and glutamine, a greater fraction of root N was derived from organic than inorganic N. Some studies also 74 reported that plants supplied with organic N show different root morphology and higher root:shoot ratio than those 75 supplied with inorganic N (8). A relation between amino acid composition and N supply was also found in legumes. We 76 demonstrated for Trifolium repens that N supplementation (ON vs. ON + IN) affected the abundance of amino acids in 77 the shoots, whereas in the roots, only the concentration (10 μM vs. 1 mM) influenced the amino acid profile (3). This 78 study indicated that root metabolism is more sensitive to nutrient quantity than quality. Thus understanding how the co-4 79 occurrence of different N forms in soil solution affects legume root and shoot performance would be fundamental not 80 only from a pure scientific perspective, but also for the productivity and quality of forage legumes in agroecosystems.

81
A complex interaction between the organic and inorganic N suggests that the co-occurrence of different N forms, rather 82 than the presence of one, affects plant N uptake. However, so far this hypothesis has not been tested for legumes.

83
Moreover, despite the extensive collection of data, studies mostly determined the uptake of amino acids from a one 84 defined N mixture (12, 13, 17), while in soil solution inorganic and organic N occur in mixtures at various ratios and 85 concentrations. Therefore, in this study the objective was to determine how different external N combinations influence 86 and regulate amino acid uptake in a N 2 -fixing legume, white clover (Trifolium repens, cv. Rivendel). To systematically 87 address some of the possible occurrences of organic and inorganic N in soil solution, we used an experimental space of 88 N treatments. Three mechanisms are proposed for the external N regulation of amino acid uptake (Fig 1a):         transitions with reference standard compounds (S1 Table). The compounds were analyzed using Sciex Analyst 1.6.2 156 software. Calibration curves (0.001-1 pmol μL −1 ) of the unlabeled standards were prepared, and the peak area of each  concentrations, 13 C 4 -Asn uptake, total N and C. The data were analyzed using R Studio 3.1.1.

178
We did not find evidence that the obtained results were related to the effect of different ratios of N 179 forms (S2 Table)

181
General clover performance 8 182 Clover was actively fixing N 2 in all the treatments with the majority of clover N obtained from N 2 -183 fixation (i.e. %Ndfa ranging from 89-97%) (S3 Table), but no significant changes in biomass, root-shoot ratio, total N 184 and C were found (S4-S7 Tables).

concentration in shoots (µmol g -1 DW) of white clover.
Uptake rate (nmol g -1 root DW) Asn in roots (µmol g -1 DW) Asn in shoots (µmol g -1 DW)  shoots, respectively, with the strongest correlation found for shoots (R = 0.83) (Fig 3).   total N dose were revealed. In the shoots, the groups related to the lowest IN (Fig 4a) and Total N (Fig S2b) 247 doses were characterized by higher concentrations of most the non-essential amino acids: Asn, Asp, Glu, Gln,

248
Cys, Pro, Gly and Ala, as well as 13 C 4 -Asn uptake rate, whereas the groups related to the higher IN and total N 249 doses contained more of the essential amino acids: Thr, Val, Ile, Leu, Phe, Tyr, Trp and Met. The same pattern 250 was shown in the roots (S2a Fig, S3 Fig). No separation related to the ON was observed (Fig 4b).  Table). Therefore, it is more likely that NO 3uptake was increased with increasing external IN and total N  (Table 4). However, we did not see a greater Asn accumulation in the roots, which were sampled 297 together with nodules, nor did we observe a decrease in the N 2 -fixation. This result is unusual because a negative ON doses, which points to that the two are connected. Although we cannot here deduce whether uptake rate 307 controls internal concentration or vice versa.

Internal amino acid composition is affected by external N doses 309
In parallel to 13 C 4 -Asn uptake rate and internal Asn concentration, external IN and total N doses 310 changed the concentrations of amino acids in the roots and shoots. At low N, we found a dominance of non-311 essential amino acids including Asn, whereas increasing external N changed the amino acid profile towards 312 essential amino acids (Fig 4). At the same time, the total N content remained unchanged (S7 Table). This is in decline of the non-essential amino acids was due to deficiency of carbon skeletons for the assimilation of NH 4 + .

319
Perhaps a decreased content of Asn in our study (Tables 3 and 4) could also be linked to its metabolization, so 320 that Asn carbon skeletons could be precursors for the synthesis of other amino acids. In that context our findings 321 support that not only the amount of amino acids, but also information on the composition of amino acids is 322 needed to determine to what extent the plant is N stressed and how the plant signals N demand between root and 323 shoot. Thus, our findings demonstrate that the external organic and inorganic N affects the accumulation of 324 certain amino acids in clover, which could help in further understanding how the plant senses various N stress 325 conditions and circulates amino acids between roots and shoots (18, 32). Soil inorganic and organic N status 326 could therefore be used as an indicator for nutritional quality of protein content in forage legumes. Furthermore,

327
our results on shoot and root amino acid composition could also be relevant in understanding how legumes 328 regulate N 2 -fixation activity as e.g. Glu, Gln, and Pro (33-35) have been found related to the N 2 -fixation 329 regulation in addition to Asn. Indeed, we found that Glu, Gln, and Pro were all related to both Asn and 13 C 4 -Asn 330 uptake rate at the low external N doses (Fig 4), which would be the conditions where we would expect the