Review Article - Journal of Natural Product and Plant Resources ( 2018) Volume 8, Issue 1
Natural products are obtained from plants sources for maintaining human health, especially in the last decade,
with more precise studies for natural therapies. Now a day, phytochemicals are used for pharmaceutical purpose has
regularly increased in many countries. Varieties of drugs are obtained from medicinal plants as reported by World
Health Organization (WHO). In the developed countries near about 80% of the total population use the traditional
medicine (medicines that are obtained from medicinal plants). Investigation has been focused on scientific evaluation
of traditional medicines for the management of various diseases, the drugs that are obtained from plant origin as
reported in ancient times. C. procera is small, erect and compact shrub, which is used in several traditional
medicines to cure various diseases. As reported C. procera has known to possess anti-cancerous, anti-inflammatory,
anti-microbial, anti-diarrheal, anthelmintic, antifertility, wound healing, antimalarial, analgesic, anti-hyperglycemic,
anti-coccidial and antipyretic activity. Different phyto constituents obtained from plants are used in many therapeutic
applications and the plant is a gift for human kind by nature. The present review discusses the uses of C. procera in
health care management.
Alfalfa, Water Stress, Endogenous nitrogen, 15N labelling, N flows
The drought presents the major environmental factor which limits the productivity and the stability of plants by affecting their growth negatively. The agronomic search contributed largely to the capacity of the world to produce more foods on limited surfaces since about forty years, thanks to the production of varieties in high yield and more resistant to the drought .
The drought effects on plants were well studied for many years, and the changes induced by the insufficiency of water supply were examined on the whole plant and its biochemical and molecular compounds as well as on the level of the different population [2,3].
Perennial plants accumulate organic reserves to initiate growth in spring in temperate regions, or when plants are recovering from other environmental stresses, they adapt to these stresses by different mechanisms, as changes in morphological, physiological and biochemical processes .
Alfalfa (Medicago sativa L.) is the most-grown forage legume in temperate regions . Being cultivated on a large part of the globe, alfalfa is exposed to rigorous climates against which it presents a remarkable capacity to resist as much the cold as the heat, and thus the drought . The water stress affects the shoot growth of alfalfa adapted to the Mediterranean climate, which come along with a greater accumulation of nitrogenous compounds in the root tissues in comparison to well irrigated alfalfa . When root growth and water uptake are prevented in dry soil, alfalfa can withstand water deficit by reducing  or stopping its vegetative growth. So, the components of taproot must specifically provide energy and the nitrogen compounds to allow the regrowth after water deficit correction.
The purpose of the present study was to define the behaviour of four varieties of alfalfa, exposed to various treatments of "drought-recovery", in term of management of endogenous N by using the 15N isotopic tracer to determine the N quantities and calculate the flows of endogenous N remobilization between the different compartments of the plant. The studied varieties were: “Europe” from France representing the oceanic climate  and 3 varieties adapted to the Mediterranean climate; “Tafilalet” from Morocco, “Tierra de Campos” from Spain  and “Moapa” from the United States of America and cultivated often in Morocco .
Alfalfa seeds of the four genotypes were sterilized and were sown on a synthetic substrate during 15 days (Oasis pinpot growing medium, Agrimedia, France). The sowing was regularly irrigated with a nutrient solution, (composition in macro and micro-elements) containing; 0.15 mM K2HPO4, 0.4 mM KH2PO4, 1 mM K2SO4, 3 mM CaCl2, 0.5 mM MgSO4, 0.2 mM Fe-Na EDTA, 14 mM H3BO3, 5 mM MnSO4, 3 mM ZnSO4, 0.7 mm CuSO4, 0.7 mm (NH4)6Mo7O2 and 0.1 mm CoCl2 . Nitrogen was added to the nutrient solution (3 mM KNO3) to repress nodule formation. When the primary trifoliate leaves appeared, seedlings were transplanted into plastic pots (two plants per pot) filled with a mixture of perlite-vermiculite (1:1, v/v). During this pre-culture, realized in greenhouse with a thermo period (20˚C/day and 18˚C/night) and a photoperiod of 16 h, plants were marked in the 15N by bringing 3 mM of K15NO3 to obtain a homogeneous labelling of all the tissues and the various nitrogenous reserves.
Treatments application and biomass determination
After one month, the 15N labelling was stopped and the different treatments were applied according to the protocol described in the Figure 1.
Figure 1: Experimental design used to determine the effect of drought (suppression of irrigation for 7, 14 or 21 days of drought) and subsequent recovery (7 days under well-irrigated conditions applied after 7 or 14 days of drought). The 15N-labeling is carried out after 15 days of sowing until the date of the first harvest (D0), i.e., during a period of 30 days for homogeneous labeling of the alfalfa tissues.
Drought treatments were imposed by withholding water  for 7, 14 or 21 days (d). After each drought period the plants were rewatered over 7 d (recovery period) (Figure 1).
During the drought period, 10 g of slow released fertilizer (10N: 11P: 18K, Osmocote, Nutri-Tabs, KB) were added to the perlite-vermiculite mixture for all the plants, to avoid differences of nutrients contribution between the control and stressed plants. At every date of harvest, 3 biological repetitions were collected for each treatment. The plant root system was carefully separated from the substrate and then was rinsed with osmosed water. The plants were then separated into leaves; steams and roots (taproot and lateral roots) and all parameters were measured on three replicates of each cultivar.
After each harvest, the various fresh organs were weighed and an aliquot of 1 g was lyophilized then was weighed to determine the dry matter biomass.
Determination of total N quantities and remobilized N fluxes
Determination of total N and analysis of 15N/14N isotopic ratio
The various lyophilized tissues were grounded into fine powder for the determination of their total N and 15N content and for the extraction and determination of the various parameters.
The system used for the isotopic analyses is an EA-IRMS (Elementar Analyzer - Isotope Ratio Mass Spectrometer) includes an elementary analyzer C/N/S (EA3000, Eurovector, Milan, Italy) coupled to a mass spectrometer of isotopic report (IRMS brand Isoprime, GV instrument, Manchester, the United Kingdom) to determine the N contents and the level of tissues 15N labelling.
Methods of calculation of N,15N quantities and N flows
The 15N labelling protocol was chosen to determine the internal remobilization processes of the endogenous N within the plant in response to water stress, followed by a rehydration period (recovery treatment).
For mass nitrogen (15N), the abundance (AN) was directly given by the mass spectrometer. It corresponds to:
AN=(15N × 100)/(14N+15N)
With 15N=quantity of nitrogen 15 and 14N=quantity of nitrogen 14 .
For a given organ, the quantity of total nitrogen qNtot by plant was given by:
QNtot=(% N × DM)/100
With % N=Percentage of nitrogen in dry matter;
DM=Dry matter of an organ expressed in mg per plant.
The method of flow calculation is the one used previously by Avice et al. . The absorbed 15N quantity by each organ was corrected according to the average of the total absorbed 15N by the whole plant and was determined from all biological repeats (n=24 plants). Any 15N remobilization was proportional to the N remobilization (15N and 14N).
• Organs called "sources" are characterized by the disappearance of a quantity of tracer for a duration Δt of the treatment.
RNs=(15Nt0 – 15Nt0+Δt) × (Nt0+Δt/15Nt0+Δt) (1)
With RNs=Remobilized N in Source Organs,
With 15Nt0=15N quantity present in the considered organ at the end of the contact period with the tracer,
15Nt0+Δt=15N quantity present in the considered organ at the time Δt of treatment,
And Nt0+Δt=Quantity of total N (14N et 15N) present in the considered organ at the time Δt of treatment.
• Organs called "sinks" are defined by an increase in their 15N content for a duration Δt of the treatment.
RNsk=ΣNRs × (15N t0+Δt/Σ15N t0+Δt) (2)
With RNsk=Remobilized N in Sink Organs,
Σ RNs=Sum of the Remobilized N in Source Organs,
15Nt0+Δt=15N quantity present in the considered organ at the time Δt of treatment,
And Σ 15Nt0+Δt=Sum of 15N quantities present in the considered organ at the time Δt of treatment.
Statistical analysis was executed by using the SPSS 12.0 software (SPSS, Chicago, HIM (IT), United States). The data were subjected to a simple analysis of variance (ANOVA) to determine significant differences between treatments. T-test was applied to verify if there were significant differences between the treatments means. The results were considered as significant in p 0.05.
Effect of a water stress treatment on the evolution of nitrogen reserves
Evolution of dry mater
On day 0, the plants dry matter (DM) of the 4 genotypes showed no significant difference in their total biomass as shown in Figure 2A. It can thus be said that on day 0, the 4 genotypes presented a biomass or a distribution of biomass, between the aerial and radical parts, which was comparable.
Figure 2: Total dry matter observed at D0 (45 days after sowing) (A) and effect of 7 days (B) 14 days (C) and 21 days (D) of water stress and rehydration period (Recovery of 7 days after each drought period) on the evolution of total dry matter in the 4 studied alfalfa varieties (TA: Tafilalet; TC: Tierra de Campos; EU: Europe; MO: Moapa). Control plants correspond to well-fed plants. The vertical bars represent the standard error for n=3. Bars with different letters are significant at 5% level of significance.
A 7 days stress did not cause a significant decrease in the total DM of the 4 genotypes, compared to the control plants values. On the other hand, one week of rehydration significantly increased their total DM compared to control and drought values (Figure 2B).
Moreover, a 14 days stress induced a significant decrease in all the studied varieties, with a resumption of growth in TA, TC and EU which reached their control values after one week recovery, which was not the case for MO (Figure 2C).
Compared to the control plants, 21 days of drought caused a higher reduction in the total dry matter in the 4 studied cultivars. For example, TC which presented the highest DM value; 6.06 (g. plant-1) in well-irrigated condition, passed to 2.28 (g. plant-1) for stressed plants (Figure 2D). After the 7 days recovery, TA and TC kept stable values in DM, while EU and MO increased them, but their control values were never reached (Figure 2D).
Evolution of the quantities of remobilized N
In control plants, the remobilized N quantities within the plant increased slightly during the first 7 days and then remained stable until the 14th day. However, it appeared that the leaves constituted the major source organ by providing about 1 mg of N (TA, TC and MO; Figures 3A1, 3B1 and 4B1) to approximately 2.5 mg (case of EU, Figure 4A1) for the benefit of roots and stems (TA, TC and EU) or only roots (MO).
Figure 3: Effects of water stress treatments (S: 7, 14 and 21 days) and a recovery period (R 21-28; rehydration of 7 days after 21 days of drought) (A2 and B2) on the evolution of endogenous N distribution between the different organs (leaves, stems, roots) among varieties: A) Tafilalet, B) Tierra de Campos. The control plants (C); (A1 et B1) correspond to plants well fed with water. The values represent the mean ± standard error for n=3.
The same results were observed in TA, TC and MO during the first 14 days of drought, suggesting that a moderate water stress period did not affect source-sink relationships between aerial and root organs towards endogenous N. On the other side, EU reacted differently, in comparison with the control plants (EU, Figure 4A1), the remobilized N quantities were almost nil during the first 7 days of stress (Figure 4A2). Then, between 7 and 14 d, water stress leaded to a strong remobilization of the foliar N in favour of the roots and stems, as in the control plants (Figures 4A1 and 4A2).
Figure 4: Effects of water stress treatments (S: 7, 14 and 21 days) and a recovery period (R 21-28; rehydration of 7 days after 21 days of drought) (A2 and B2) on the evolution of endogenous N distribution between the different organs (leaves, stems, roots) among varieties: A) Europe, B) Moapa. The control plants (C); (A1 and B1) correspond to plants well fed with water. The values represent the mean ± standard error for n=3.
In the control plants and regardless of the considered genotype, the source-sink relationships for the remobilized N observed between 0 and 14 days were completely changed between 14 and 21 days. After 14 days, the leaves became major sink organs by incorporating high amounts of remobilized N from the stems (TA, Figure 3A2), roots (TC, Figure 3B2) or the two organs in the case of EU and MO (Figures 4A2 and 4B2).
Between 14 and 21 days, the source-sink status for endogenous N was profoundly altered in response to water stress compared to control plants. Effectively, after 14 days of drought, the leaves became major source organs by remobilizing large amounts of N mainly towards the roots and secondarily towards the stems (Figures 3A2, 3B2, 4A2 and 4B2).
The strongest foliar N remobilization to other organs was observed in TA, with 13, 31 (mg N.plant-1) remobilized from leaves at the 21 days of drought (Figure 3A2).
On the other hand, compared to a 21 days water stress, a week of soil hydric status recovery caused a great change in source-sink relationships, which was translated by an important remobilization of endogenous N from roots and stems in favour of the leaves, with a higher amount of remobilized N than that observed in stressed plant leaves. These observations were verified among the 4 studied varieties (Figures 3A2, 3B2, 4A2 and 4B2).
The distribution of the marking 15N (distribution of the endogenous nitrogen)
Since the previous results demonstrated that all genotypes showed a strong N remobilization after 21 days of drought (Figures 3 and 4), we presented in Table 1 the distribution of 15N (in % of total 15N from the labelling), which resulted from its distribution between the different organs (leaves, stems and roots) for the 3 treatments and for the 4 alfalfa genotypes.
|Organ||Treatment||Tafilalet||Tierra de Campos||Europe||Moapa|
|Leaves||Control||58.5 ± 0.5 a||54.7 ± 7.1 a||50.0 ± 11.5 a||55.2 ± 2.3 a|
|Drought||19.9 ± 5.4 b||28.3 ± 1.1 b||36.8 ± 1.7 b||28.8 ± 6.7 b|
|Recovery||48.5 ± 0.7 b||55.7 ± 2.6 a||50.9 ± 4.4 a||48.6 ± 5.6 a|
|Stems||Control||16.9 ± 1.4 b||20.3 ± 2.7 ab||21.7 ± 4.7 a||20.4 ± 3.1 a|
|Drought||32.4 ± 3.2 a||27.5 ± 0.1 a||22.6 ± 2.4 a||29.5 ± 4.3 a|
|Recovery||22.8 ± 2.9 b||19.1 ± 2.7 b||21.6± 2.3 a||22.1 ± 2.5 a|
|Roots||Control||24.6 ± 1.1 b||25.0 ± 4.5 b||28.3 ± 7.4 b||24.4 ± 2.7 b|
|Drought||47.7 ± 5.2 a||44.2 ± 1.2 a||40.6 ± 2.7 a||41.7 ± 2.8 a|
|Recovery||28.7 ± 2.3 b||25.2 ± 3.2 b||27.5 ± 2.8 b||29.3 ± 3.5 b|
Table 1: Effects of 21 days treatment (recovery of 7 days after 14 days of water stress) on the evolution of the 15N distribution (in % of the total 15N) between the different organs (leaves, stems and roots) in 4 varieties of alfalfa. The values represent the mean ± standard error for n=3.
In comparison with the control plants, the percentage of 15N decreased in the leaves during the drought period for all genotypes. The most significant reduction was observed in TA with a 38.6% drop between control and stressed plants.
Drought significantly increased the percentage of total 15N in stems in TA (an increase of 15.5%), while it did not affect the other genotypes (TC, EU and MO). In roots, 15N significantly increased during drought in TA (+23.1%), TC (+19.2%), EU (+12.3%) and MO (+17.3%). Compared to the leaves of the control plants, a one week recovery period allowed all genotypes to regain their 15N percentage of the control. In the stems, the recovery period did not cause a change in 15N percentage compared to control plant stems. In the roots, the rehydration allowed all the genotypes to reach their 15N level of the control (Tables 1 and 2).
|Treatment||Organ||Tafilalet||Tierra de Campos||Europe||Moapa|
Table 2: Statistical comparison on the evolution of 15N repartition (in percentage of total 15N) among different organs (leaves, stems and roots), for each treatment and each studied variety of alfalfa. The values represent the mean ± standard error for n=3
The objective of this work was to determine if the responses of alfalfa, in terms of nitrogen management, to the application of water stress (whether or not followed by a period of recovery) could help to better tolerate drought and/or promote the resumption of aerial growth; when the water status became favourable again. This study was carried out in four alfalfa genotypes, 3 genotypes from the Mediterranean (Tierra de campos, TC, Ecotype Tafilalet, TA and Moapa, MO) and one oceanic (cv. Europe, EU), with the aim to observe behavioural differences in terms of biomass and management of endogenous nitrogen (storage and remobilization of nitrogen reserves) against an increasing intensity of water stress.
According to the obtained results, it appeared that only a severe drought period (of 21 days) could cause a large drop in the total dry matter biomass of all the studied varieties, which despite a week of rehydration did not occur to recover their growth levels from control plants and with regard to plants under low and moderate drought treatments (7 and 14 days of drought). These results were similar to those obtained by Kimani et al. , who showed that severe water stress reduces biomass in the Angola pea legume, of 34 to 54%.
Other similar studies have also shown that plants under water stress conditions often reduce the production of their biomass [7,16-18], as an adaptation trait of drought resistance.
A parallel study was conducted by Erice et al. , on other parameters, studying the DM of each organ and demonstrating that after moderate water stress, TA and MO decreased leaf DM, which may help to maintain relative water content (RWC) in these varieties. In contrast, roots were not affected by water deficiency, showing less sensitivity to drought. After moderate stress, one week recovery increased root DM in TA, which may allow higher water soil prospecting. Also a severe water stress of 21 days did not allow discrimination between varieties in relation to DM and water status parameters. Severe drought (21 days of water withholding) reduced leaf and stem DM with no differences among varieties. Nevertheless a severe drought did not affect root DM, except for TC, which showed decreased root DM due to its highest control value. Root DM, in contrast to that observed for leaves, was not affected by drought, confirming that roots are generally less affected than are shoots [16-21].
So we could suggest that the maintenance of total biomass was mainly related to root growth and to a lesser extent to leaves and stems.
The effect of water stress on endogenous nitrogen redistribution (stored in the different organs of the plant prior to the start of treatments), was then studied by 15N labelling and showed a significant decrease of N quantities present in the leaves of plants subjected to water stress. In drought conditions, leaves therefore became major N source organs. EU, the oceanic variety reacted upon the application of 14 days of stress by remobilizing its N reserves towards the roots unlike the other varieties, which reacted only after a severe stress of 21 days, suggesting that EU was more sensitive to drought than the Mediterranean studied varieties.
The strength of a source organ is described as being the net rate at which a substance is transported (or stored inside) of that organ .
After an analysis of the endogenous nitrogen kinetics, it was found that the foliar nitrogen reserves were mainly remobilized towards the roots and lesser towards the stems, with no differences among varieties. All of these observations suggested that, in response to prolonged water stress, alfalfa was able to store in its roots the remobilized N from aerial tissues.
The increase of root biomass was also a means to increase the N storage in alfalfa as shown [7,21,22]. This ability to store organic reserves, under stressful conditions, was crucial for N conservation, which could be remobilized to support growth when soil water status became more favourable.
The work realized by Ourry et al. , under optimum culture conditions, showed that the most important aerial growth is obtained in alfalfa with the highest root N content.
Globally, 15N labelling suggested that during the drought, the nitrogen stored in the leaves decreased and was remobilized to the roots and stems for TA and only to the roots for TC, EU and MO. In case of return to a favourable water conditions, the plants of the 4 genotypes were able to redistribute this nitrogen from the roots (and stems) to the leaves. It appeared, therefore, that the redistribution of leaf nitrogen to the reserves tissues (roots and to a lesser extent the stems) was rather a general response to water stress which was observed in all the studied alfalfa genotypes.
As explained by Ourry et al. , the recycling N within the plant allowed its constant use by different tissues and at various stages of development. However, the redistribution amplitude of foliar nitrogen appeared to vary from one genotype to another. Indeed, TA was the genotype for which the quantities of remobilized N were the most important after 21 days of drought, while TC had the lowest amounts. At TA, this greater ability to redistribute N compounds; previously stored at the foliar level towards roots and stems, could be an advantage, when soil moisture status became favourable, by allowing this genotype to use these N reserves to support N requirements of growing leaves.
The evolution of 15N quantities resulting from the labelling period (carried out 30 days before the beginning of the treatments; Figure 1) made it possible to estimate the effects of the drought and the recovery period on the endogenous N redistribution (stored in the different organs before the start of treatments).
The results shown that for all genotypes, the leaves contained a high N quantity when plants were well hydrated. Consequently, they were potentially presenting an important source organ of N in water stress case, since their N levels had been found to be greatly reduced compared to the control plants. Indeed, the 15N quantities doubled in the taproot of the stressed plants compared to the control. Nevertheless, water stress was translated by a decrease of the taproot N contents, suggesting that there was a modification in the chemical composition of the different nitrogen fractions; such as amino acids and soluble proteins which are the most important forms of storage.
It was shown that the roots biomass was maintained in case of water stress  and that the endogenous N was strongly redistributed towards roots. Given that the roots represented the major organic reserves organ, it was particularly interesting to verify if the pivot was capable of storing temporarily the redistributed N by the aerial parts in response to drought. To answer to this question and others, we have to intensify the research for the various N reserves in the root of the various genotypes, as well as to study other physiological and biochemical approaches of alfalfa, which may help for drought-tolerant cultivar improvement.