Vandana Kushawaha·Satya Prasad Chaukiyal

Abstract Morphology, biomass, nitrate reductase (NR)and nitrogenase activity in Albizia chinensis(Osb.)Merr.nodules were assessed on monthly and seasonal basis for 1 year.Average NR and nitrogenase activity was higher during the rainy season,reaching a maximum in August.Thereafter,activity decreased through autumn and reached a minimum value during winter.Fresh and dry biomass of nodules increased gradually from summer to the rainy season and then started decreasing with the onset of winter as nodules began to senesce.Among four developmental stages of the nodules that correspond to their increasing age,NR and nitrogenase activity remained low in stage 1 nodules and peaked in stage 2. The activity of both enzymes further decreased with increasing age(stage 2 to stage 4).Morphological features such as shape,diameter and color varied considerably among the developmental stages.Stage 1 nodules were cream-colored,oval to heartshaped with smallest average diameter whereas at stage 2,they became bilobed to tetralobed.On the other hand,stage 3 nodules had the largest average diameter and were multilobed in structure.Stage 4 nodules that correspond to the senescing stage were dark brown to black,multilobed,flattened and hollow due to degeneration of nodular tissue.

Keywords Nitrogenase activity·Nitrate reductase(NR)activity·Nodule biomass·Nodule morphology·Nodule senescence

Introduction

The world population has increased by 78%since 1978 and has largely relied on nitrogen-based chemical fertilizers to meet increasing food demands(Galloway et al.2004)but at huge economic and environmental costs.Of the total fertilizer applied,less than 30%is estimated to be used by crops and the rest is lost to the environment through denitrification, volatilization or leaching, causing serious hazards(Cheng 2008).It is therefore important to achieve synchrony between nitrogen supply and demand by crops without degrading environmental qualities. Biological nitrogen fixation is an environment-friendly way of improving soil nitrogen status.With increasing concern for environment and food security,the study of biological nitrogen fixation is needed more than ever.The use of nitrogen-fixing species in forestry and agricultural practices is therefore a sustainable method, especially in countries such as India where protection and restoration of soil fertility is a critical problem.

Nitrogen metabolism of a plant can be assessed by studying enzymes involved in nitrogen fixation and assimilation.During biological nitrogen fixation,the symbiotic association of legumes with Rhizobium results in the formation of root nodules where atmospheric nitrogen is reduced to ammonium by an oxygen-sensitive enzyme,nitrogenase(Hirsch 1992).Legumes can also fulfill their nitrogen requirement through nitrate uptake from soil.Nitrate once taken up by the plant is reduced to nitrite by nitrate reductase(NR)and then to ammonium by nitrite reductase(NiR).Because NR catalyses the rate-limiting step of the nitrate reduction pathway,its activity is used to assess nitrate uptake and the assimilation potential of a plant.Ammonium produced either by biological nitrogen fixation or through nitrate reduction is incorporated into amino acids and proteins(Li and Oaks 1995;Huber et al.1996;Crawford et al.2000).

Water stress and temperature are among the most important factors influencing activity of nodules(Serraj and Adu-Gyamfi 2004).Therefore nodular activity varies greatly during nodule development as the seasons change.Moisture deficiency and extreme temperatures limit rhizobial growth and may even lead to complete failure of nodulation.Defoliation,drought and temperature stress may also result in premature senescence in nodules(Witty and Minchin 1988;Puppo et al.2005).However,developmental senescence is age dependent and usually starts in 5-to 11-week-old seedlings.The process is relatively slow and well regulated.Senescence of nodules eventually leads to their death and decay followed by release of large amounts of nitrogen into the soil.Therefore,higher the nitrogen fixation potential of a plant,greater the amount of nitrogen released.

In the present study,variation in nitrogenase and NR activity in Albizia chinensis nodules with respect to different developmental stages and seasons were assessed.Seasonal variation in biomass production of the nodules was also recorded because it is directly correlated to activity of the nodule.In addition,changes in morphological variables of the nodule during its entire life span were studied.Results obtained in this study will provide an important set of information about nitrogen metabolism,biomass and morphology of the A.chinensis nodules for future studies to improve its nitrogen fixation potential.

Materials and methods

Nursery-grown,potted A.chinensis seedlings at the Plant Physiology Discipline,Forest Research Institute,Dehradun,India were used.The study was carried out for 1 year(2015-2016)which was divided into three seasons:summer(March,April,May and June),rainy(July,August,September and October)and winter(November,December,January and February).After washing and blot-drying the freshly harvested nodules,NR activity,nitrogenase activity and biomass of the nodules were measured at monthly intervals.Variation in morphology and activity of the nodules at different developmental stages was also studied.Results were analyzed on monthly and seasonal basis.

In vivo NR activity

In vivo NR activity was assessed as described by Klepper et al.(1971).Freshly harvested nodules were weighed(0.5 g)and transferred to an incubation media(0.15 M KNO3and 0.10 M KH2PO4buffer,pH 7.6)in test tubes.Potassium nitrate releases nitrate ions,which acts as a substrate for the NR enzyme.Samples were first vacuuminfiltrated for 3-4 min followed by incubation in the dark at 30°C for 1 h in a shaking water bath.They were then transferred to a boiling water bath for 4-5 min to inactivate the enzymes.The nitrite accumulated in nodules as a result of NR activity thus effectively diffuses into the surrounding media.

Nitrite was then quantified by the method of Evans and Nason(1953).The required amount of sample was pipetted into a test tube,then 1 mL sulphanilamide and 1 mL 0.01%1-naphthyl ethylene diamine-dihydrochloride was added.These sample mixtures were kept at room temperature for 25 min for color development;thereafter,the final volume was brought to 6 mL using distilled water.Absorbance recorded at 540 nm in Spectrascan UV-2700 spectrometer(Chemito,Mumbai,India)were used to calculate the NR activity in each sample.

Nitrogenase activity

Nitrogenase activity was estimated using the acetylene reduction assay of Hardy et al.(1968).Nitrogenase can reduce acetylene(C2H2)to ethylene(C2H4)in a reaction similar to reduction of nitrogen to ammonium.Therefore in this assay,acetylene is provided instead of nitrogen as substrate of nitrogenase.One gram of nodule was taken in air-tight incubation vials and 10%of the air in the vials was replaced with an equal volume of acetylene.The nodule samples were incubated in a shaking water bath for 1 h at 30°C in the dark and then transferred to an ice-filled container to stop the reaction.Ethylene is then quantified using Nucon(Delhi,India)5765 Gas Chromatograph.The amount of ethylene released is directly proportional to nitrogenase activity.

Biomass study

Total nodules from individual plants were weighed immediately after harvesting,washing and blot drying to obtain fresh weight(FW).Nodules were then oven dried at 70°C until a daily weighing showed that the weight was constant and moisture was absent.The final constant value was recorded as dry weight(DW).

Study of nodular stages

In a companion experiment,shape and size of nodules at different developmental stages were observed to categorize the nodules into four stages(Fig.1a-d):1,newly emerging;2,young;3,mature;and 4,decaying.Shape,color and diameter of each nodule in the different categories were recorded.Nitrogenase activity and NR activity were also assessed for different stages following the previously mentioned methods.

Statistical analyses

The data was subjected to statistical analyses using the software ‘Genstat, Release 3.2' (?Lawes Agricultural trust,IACR-Rothamsted).Monthly values for NR activity,nitrogenase activity and biomass (FW and DW) were analyzed through one-way ANOVA.Least significant difference(LSD)was obtained for given sources of variation at 5%level of significance.

Results

Variation in morphology of different stages of nodules

Fig.1 Nodules of four developmental stages from Albizia chinensis seedlings.a Stage 1:newly emerging,b stage 2:young,c stage 3:mature,d stage 4:decaying

Stage 1 nodules of A.chinensis which first appeared in May,were oval to heart-shaped,cream-colored and had an average diameter of(2.50±0.07)mm.Young nodules(stage 2)were comparatively larger,bi-to tetra-lobed,cream-colored at the distal end and brownish white on the outside but pink inside at the proximal end with average diameter of (4.48±0.12) mm. Nodules attained their maximum size at stage 3(mature)with an average diameter of(9.55±0.49)mm and were multi-lobed,forming large clusters.Proximal ends were dark brown,indicating loss of legheamoglobin and the distal end was cream-colored to light brown superficially but pink inside,representing the active zone.In winter,they gradually started degenerating and by the end of the season,they were hollow,shrunken and dark brown to black.These nodules were categorized as stage 4 nodules and the average diameter was(5.95±0.24)mm(Table 1).

Variation in nitrogenase and NR activity in nodules at different stages

Activity of both the enzymes greatly increased from stage 1 to stage 2 and then decreased from stage 2 to stage 4 nodules. Nitrogenase activity was recorded maximum(11,510.93±207.15 nmol C2H2reduced g-1h-1)and minimum(195.03±72.56 nmol C2H2reduced g-1h-1)in stage 2 and stage 4 respectively.Contribution of stage 2 nodules to total nitrogenase activity was 61%and that of stage 4 was 1%(Fig.2a).Similarly,NR activity was found maximum(485.11±12.33 nmol NO3-reduced g-1h-1)in stage 2 and minimum (50.21±5.96 nmol NO3-reduced g-1h-1)in stage 4 nodules that accounts for 54%and 6%of the total NR activity respectively(Fig.2b).

Seasonal variation in nitrogenase and NR activity

Nitrogenase activity was highest in the peak rainy season i.e. August (18,709.12±1954.60 nmol C2H2reduced g-1h-1)and lowest in January(24.79±8.27 nmol C2H2reduced g-1h-1).No nodules were observed during March and April.Similarly,NR activity increased gradually from May onwards,peaked in August(571.28±20.71 nmol NO3-reduced g-1h-1)and decreased thereafter until the end of the winter; hence, the lowest activity was in February (67.02±1.93 nmol NO3-reduced g-1h-1).Differences among monthly activities of nitrogenase and NR were statistically significant at 5%level of significance(Table 2).On a seasonal basis,nitrogenase and NR activities were highest during the rainy and lowest in winter season(Fig.3a,b).

Seasonal variation in nodule biomass

Fresh and dry weight were maximum in August(FW:11.68±2.08 g, DW: 2.51±0.23 g) and minimum inMay(FW:0.57±0.07 g and DW:0.08±0.01 g).Differences among monthly means for fresh and dry weight were significant at 5%level(Table 2).Among seasons,highest biomass was observed in rainy season followed by winter and lowest in summer(Fig.3c).

Table 1 Variation in morphological features at different developmental stages of Albizia chinensis nodules

Fig.2 Percentage contribution of nitrogenase(a)and nitrate reductase(b)activity at various developmental stages of Albizia chinensis nodules

Table 2 Monthly variation in nitrogenase activity,NR activity and biomass of Albizia chinensis nodules

Fig.3 Seasonal variation in nitrogenase activity(a),nitrate reductase activity(b)and biomass(c)of Albizia chinensis nodules.FW is fresh weight;DW is dry weight

Discussion

In A.chinensis seedlings,nodulation started in May when the plants received first rain shower along with increased temperature. During this month, NR and nitrogenase activity were low,which might be primarily attributed to the presence of only stage 1 nodules that have relatively lower activity of the two enzymes.Moreover,summer imposes water and high temperature stress that might have significantly reduced the enzymatic activity(Hungria and Vargas 2000).Hungria and Franco(1993)also reported an inhibiting effect of high temperature on nitrogen fixation in Phaseolus vulgaris. Moisture deficiency in soil during summer may cause carbon shortage,changes in oxygen permeability and interruption in feedback pathway which altogether reduces nodule activity(Serraj et al.1999).

During rainy season,the seedlings comprised stage 1 and 2 with a small amount of stage 3 nodules.As the number of stage 2 nodules(most active)increased,NR and nitrogenase activity also increased gradually and peaked in August.Young nodules are high in leghemoglobin and therefore more active than newly formed and the older ones(Sharma and Ambast 1984;Chaukiyal 2007).Nitrogen fixation is usually optimum after 4 weeks of infection and lasts for a very short time(Bethlenfalvay and Phillips 1977)as observed in this study.By the end of the rainy season,stage 3 nodules were more abundant indicating the start of senescence and hence activity of the enzymes dropped.Nodule senescence is a programmed phenomenon that triggers the activity of numerous proteolytic enzymes that degrade nodular proteins including leghemoglobin(Pladys and Vance 1993;Gunther et al.2007).Besides proteolysis,degradation of membrane lipids,nucleic acids and sugars is also triggered(Van de Velde et al.2006).All these activities eventually lead to disintegration of nodules.It is a slow process that facilitates nutrient remobilization from nodule to other plant parts.

During winter,activity of both the enzymes decreased further.Nodules of all the stages were observed at the beginning of the season but nodulation gradually stopped and hence stage 1 nodules were no longer present.The number of older nodules(stage 3 and 4)increased and younger nodules(stage 2)decreased.By the end of the season,only stage 4 and a few stage 3 nodules were present,which might be due to the retardation effect of low temperature on nodular activity(Gibson 1971).

We thus deduced that both high and low temperatures have negative impact on nodule activity of A.chinensis.Nitrogen fixation considerably reduced at 15°C and 35°C in soybean and was higher at moderate temperature(25°C)(Montanez et al.1995).Legheamoglobin content was also significantly reduced at two extreme temperatures i.e.21 and 36°C in Vigna sinensis(Dart and Mercer 1965).Pokhriyal et al. (2001) also reported relatively higher nitrogenase and NR activity during rainy season and lower during winter in nodules of Acacia nilotica in a mixed planting of Acacia and Eucalyptus.Similarly,nitrogenase activity was reported higher in nodules of Myrica esculenta during the rainy season and lower in winter(Chaukiyal et al.2014).

Another reason for low nitrogen metabolism in A.chinensis nodules during winter might be‘leaf senescence'.By the end of October,leaves started senescing.Genes expressed during nodule senescence have a considerable degree of homology with those expressed during leaf senescence indicating that the two processes are closely linked(Lohman et al.1994).Initiation of both leaf and nodule senescence together in the present study are probably responsible for the continual decrease in enzymatic activity during this season.Leaves provide the reduction potential to nodules required for nitrogenase activity.In addition, during leaf senescence, the concentration of reduced nitrogen compounds such as amino acids flowing down through phloem increases around the nodule,which causes feedback inhibition of nitrogenase activity(Lucinski et al.2002).Senescence of nodules in this study might have been induced by both age and stress(low temperature),leading to their rapid degeneration during winter.Similar results were obtained by Pokhriyal et al.(1991)in Dalbergia sissoo and by Chaukiyal(2007)in Pongamia pinnata. Gradual decay of nodules can be characterized by degeneration of leghemoglobin and disintegration of bacteriodes.During this process,nodule color shifts from pink(active)with leghemoglobin to green due to the breakdown of the heme group(Roponen 1970).The estimated amount of nitrogen fixed by legumes further varies with Rhizobium efficiency,host plant genotype,soil characteristics,climatic conditions and the method used to assess fixation rate.

The biomass of nodules in A.chinensis was highest when rainfall was highest(August)and lowest in winter,with a minimum value in February.Increased activity of nitrogenase and NR during the rainy season,which consequently leads to higher nitrogen metabolism,might have enhanced the overall growth of seedlings resulting in the increased biomass of the plants including nodules.Moreover during this period,most of the nodules have reached stage 2 or 3 which are relatively large and multilobed.With the onset of winter,reduced NR and nitrogenase activity along with gradual decay of the nodular tissue explains the lower biomass in winter.By the end of winter,nodules were partially or completely hollow and flattened,resulting in greatly reduced biomass.A microscopic analysis by Van de Velde et al.(2006)showed that during nodule senescence,first the microbial partner is targeted for degradation and later the plant cells of the nodule die,leading to complete decay and collapse of the nodule.

Nodule biomass and number has earlier been reported to vary with season and age of the seedling(Aryal et al.2000;Hossain et al.2001).Chaukiyal et al.(2013)also reported maximum nodule biomass during rainy season in Pongamia pinnata.

Albizia chinensis is fairly resistant to drought,high pH,salinity and poor soil quality.It also has a repellent property against subterranean termites.As we report here,it also has high nitrogen fixation and assimilation potential,further increases its value.Therefore,its use in agroforestry practices in the long term will reduce the use of nitrogen fertilizer to a greater extent.We also recommend using this species to restore highly degraded land and reclaim soil.Our results in relation to fluctuations in nitrogenase activity,NR activity and biomass with nodule age and changing seasons provide a base for research to increase its nitrogenuse efficiency,stress tolerance and productivity.

AcknowledgementsThe present study was conducted in the Plant Physiology Discipline,Forest Research Institute(FRI),Dehradun,India.We are grateful to Dr.T.C.Pokhriyal,(retired),Botany Division, FRI, Dehradun for his guidance. We also thank the two anonymous reviewers for valuable suggestions that helped to improve the manuscript.

Authors contributionVK performed the experiments,analyzed the data and wrote the manuscript;SPC designed the study,helped interpret results and improved the manuscript.

Compliance with ethical standards

Conflict of interestAuthors declare that they have no conflict of interest.