Pramod Kumar?Yogeshwar Mishra

Abstract Biochemical changes associated with flowering in Bambusa arundinacea Linn and Bambusa nutans Wall.ex Munro were analyzed.Gregarious flowering was initiated in natural areas and plantations of B.arundinacea in late 2014 and reached full bloom in early 2015,whereas sporadic flowering recorded during 2014–2015 in vegetatively propagated plants of B.nutans.Leaf and nodal shoot samples from flowering culms/shoots and nonflowering culms/shoots were collected in February 2015 at the initiation of fruit filling,then analyzed and compared for soluble sugars,phenols and peroxidase activity.In both species sugars were higher in leaves of flowering culms/shoots and lower in nodal shoots of flowering culms/shoots compared to the nonflowering.Phenols were lower in leaves and nodal shoots of flowering B.arundinacea,but higher in leaves and decreases in nodal shoots of B.nutans.Peroxidase activity increases in leaves and nodal shoots of B.arundinacea after flowering but increases in nodal shoots and decreases in leaves of B.nutans.

Keywords Sugars·Phenols·Peroxidase activity·Flowering·Bambusa arundinacea·Bambusa nutans

Introduction

Bamboos are fast-growing evergreen perennial plants of family Poaceae having immense economic importance and thus has potential as an afforestation species.About 14 million hectares of the Earth’s surface is covered by bamboos with 80%in Asia(Tewari 1992).In India,the second richest country in bamboo genetic resources following China(Bystriakova et al.2003),9.57×106ha-1,about 12.8%of the total forested area is covered by bamboo plantations(Sharma 1980).The distribution of bamboo is associated with different agroclimatic zones of India(Varmah and Bahadur1980)and influenced by human interventions (Boontawee1988)and rainfall(Gamble 1896).DendrocalamusandBambusaare the two predominant genera of bamboo in the dry tropical region of India.

Bamboo is of manifold importance in day-to-day rural and semi-urban life with crucial roles in cultural,artistic,industrial,agricultural,construction and household needs(Mukherjee et al.2010).Mature bamboo leaves contain phenolic acids,and the roots contain cyanogenic glycosides(Das et al.2012).Nearly half of the national demand of raw material for the paper and pulp industry is fulfilled by bamboos.The government through the National Mission on Bamboo Application(NMBA)launched various programmes to enhance and sustainably use bamboo resources to meet the requirement of bamboo raw material for various sectors.However,the long vegetative period and peculiar flowering behavior of bamboos hampering the availability of sufficient seeds for planting stock production;therefore,bamboo plantations are normally propagated asexually.

Reproductive growth is initiated by the onset of flowering,which is affected by various external and/or internal signal(s),leading to sexual propagation and thus fruit/seed formation.Bamboo flowers once in the life of this perennial with no signs of a flowering stimulus such as photoperiodic induction or low temperature(Zhang and Liu 2003).Bamboo always blooms and dies at the same time(Janzen 1976;Sharma 1994;Li 1997).Bamboo species have one of three types of flowering behavior annual or continuous,gregarious or periodic,and sporadic or irregular.In most of the commercial bamboos, flowering occurs synchronously in all the daughter clumps of a parent clump after a predetermined vegetative period.The flowering rotation cycle of some of the bamboo species has been compiled.Established growth cycles as well as genetic factors are responsible for bamboo flowering;however,the precise physiological mechanism and biochemical changes have yet to be explored.John and Nadgauda(2002)described the sequence of events in gregarious flowering that is initiated in September-October and ends with seed shed in December-January.Han(2003)defined the role of nutrition,hormonal adjustment and genetic expression in the flowering process.Peroxidases participate in indole-3-acetic acid catabolism and can modify the hormonal balance in plants,thusleading to the modulation of morphogenesis.

In Maharashtra,Madhya Pradesh and Chhattisgarh states of India,gregarious flowering was initiated in natural areas and plantations ofB.arundinaceain December 2014,reached a full bloom in early 2015 and seeds were shed in March-April.During 2014 and 2015,however,vegetatively propagated plants ofB.nutansgrowing in clay pots at the Tropical Forest Research Institute campus flowered sporadically.As noted earlier, flowering in bamboos is a peculiar cyclic phenomenon that occurs after a specific time interval depending on the species.The average time span between two gregarious flowerings is about 50 years forB.arundinaceaand 35 years forB.nutans.The physiological reasons for such flowering behavior in bamboos are not comprehensively defined.Because carbohydrates,antioxidants and oxidative enzymes play key roles in any morphogenic process,we quantified sugars,phenols and peroxidase activity inB.arundinaceaandB.nutans.Our present findings help characterize the flowering phenology in bamboos.

Materials and methods

Fig.1 Flowering clump of Bambusa arundinacea

Fig.2 Flowering plant of Bambusa nutans

Fig.3 Flowering shoot of Bambusa arundinacea

Fig.4 Flowering shoot of Bambusa nutans

Biochemical assays were carried out in February 2015 when flowering was at its mature phase and fruit filling had also begun in both the species(Figs.1,2,3,4).Leaf samples and nodal shoot samples were collected from flowering and nonflowering culms/shoots ofB.arundinaceaclumps in a plantation(N23°05′58.5′′E079°58′56.5′′)andB.nutansgrowing in clay pots(N23°06′01.6′′E079°59′23.1′′)at the Tropical Forest Research Institute campus in Jabalpur,India at 414 m a.s.l.with almost similar edaphic and climatic conditions.Soil of the area is reddish brown loam to clay with sand-silt-clay in 4:3:3,moderately well drained with good water-holding capacity,pH 7.5 and electrical conductivity(EC)of 156× 10-6μs.Clay pots(hemispherical volume 70.65 m3)for growing the bamboos contained a mixture of soil,sand and farmyard manure(2:1:1)pH 7.2 and EC of 151× 10-6μs.In vegetatively propagated plants ofB.nutans, flowering began in September 2014 and flowering and nonflowering shoots survived for a long time.The top three leaves and top three nodal shoot segments from flowering and nonflowering shoots of both species were collected randomly from 10 shoots in separate respective samples,chopped and mixed.Total soluble sugars(Duboiset al.1956)and phenols(Bray and Thorpe 1954)were estimated in an alcoholic extract of 0.1 g fresh tissues.Three replications for each representative sample with three subsamples of each replication were used.For estimation of total soluble sugars,1.0 mL alcoholic extract was vaporized in awater bath,then 1.0 mL distilled water,1.0 mL 5%phenol solution and 5.0 mL H2SO4were added.Absorbance of the reaction mixture at 490 nm was recorded using a UV-visible spectrophotometer(GBC,Hampshire,IL,USA)and compared with a glucose calibration curve and quantity expressed as μg/g fresh mass.Phenols were estimated using 5.0 mL ethanolicextract,1.0 mLFolin-Ciocalteureagent(50%)and 2.0 mL Na2CO3(20%w/v).The solution was heated gently in a boiling water bath,then cooled and filtered.Absorbance of the reaction mixture at 650 nm was recorded,quantified with a Catechol calibration curve and quantity expressed as μg/g fresh mass.

For measuring peroxidase activity(EC 1.11.1.7),an acetone powder formed by homogenizing 100 mg samples in chilled acetone(Mahadevan and Shridhar 1986).Acetone powder was suspended in 1-mL 0.02 M sodium phosphate buffer(pH 6.4)in an Eppendorf tube and centrifuged for 10 min at 10,000 rpm.The supernatant was used to estimate peroxidase activity using the method of Rama Rao et al.(1982).Absorbance at 470 nm was recorded every 30 s for 3 min.Soluble protein content in the enzyme extract was estimated using the method of Lowry et al.(1951).Peroxidase activity was computed in accordance with protein content and expressed as Δ470protein min-1mg-1.

Data were subjected tottest to compare variables between the means for flowering and nonflowering culms and between the two species using standard statistical procedures(Gomez and Gomez 1984)in MS Excel,WindowsXP2007.

Results

Total soluble sugars,phenols and peroxidase activity with significance of the differences between flowering and nonflowering shoots in two species are presented in Tables 1 and 2 and between the two species are presented in Table 3.Total soluble sugars were 67.16 and 14.45%higher in leaves of flowering shoots ofB.nutansandB.arundinacea,respectively,than in leaves of the respective nonflowering shoots,but 23.00 and 33.39%lower in nodal segments ofB.nutansandB.arundinacea,respectively,than in their nonflowering shoots.Phenols were 29.90%higher in leaves of flowering shoots ofB.nutansbut 25.76%lower in leaves of flowering shoots ofB.arundinaceacompared to nonflowering shoots.Phenols were 11.70 and 39.42%lower in nodal segments of flowering shoots of both species than in their nonflowering shoots.Compared with the activity in nonflowering shoots,peroxidase activity in nodal segments of flowering shoots was 205.80 and 13.70%higher inB.nutansandB.arundinaceaand 27.81%higher in leaves of flowering shoots ofB.arundinaceabut 54.98%lower in leaves of floweringB.nutans.

Discussion

In general,the mechanism responsible for flowering is a multifaceted controlled process leading to quantitative changes in compounds for metabolic processes.Bamboos flower independently of day length after attaining a certain vegetative age.This peculiar flowering behavior might be regulated by a specific microRNA and require energy that is available to the plant in the form of sugars.Our results revealed increases in soluble sugars in leaves of both species during flowering in accordance with Sachs(1977)view that abundant nutrients favor flower bud differentiation.However,Garg et al.(1998)noted significant reduction in carbohydrates at the end of flowering inD.strictus.Carbohydrates that are stored in leaves and stems translate into sugars and accumulate in apical meristematic tissues,which act as a carbon source for developing buds(Zhou and Xu 2002).Han(2003)advocated transition from quantitative to qualitative changes through network of biochemical processes during organogenesis.Similarly,Hong(2015)described key factors during flowering in twobamboo species native to China viz.Phyllostachys heterocladaandNeosinocalamus affinisand opined that at physiological maturity,carbon and nitrogen metabolism changes,along with hormonal alterations lead to an increase in soluble sugar content.After attaining optimum level for each substance,the plant will finish floral bud differentiation stage and transition into the generative stage,or undergo flowering.Sugars are utilized to provide structure and energy during flowering and thus were lower in nodal segments of flowering shoots of both bamboo species than in nonflowering shoots due to unloading from phloem cells to the sink,i.e.,seed filling.Mohammad et al.(2013)reported that the sugar trehalose-6-phosphate(T6P),which is present in plants in minute amounts,acts as a signaling molecule and aids in controlling flowering time in relation to energy reserves.However,the role of T6P as a signaling factor has not been explored in bamboos.

Table 1 Comparison of biochemical properties between flowering and nonflowering shoots of Bambusa arundinacea(two-sample t test)

Table 2 Comparison of biochemical properties between flowering and nonflowering shoots in Bambusa nutans(two-sample t test)

Phenols normally provide resistance to stresses in plants in addition to their role in various intermediary metabolic pathways.A decrease in phenols indicates utilization in expansion growth during organogenesis,i.e.,development of flowering buds.However,increased level in leaves ofB.nutansmight be due to collection of samples after a quite a long period of flowering,leading to an optimum level in flowers and thus its accumulation in leaves.This increased level further enhances resistance and may be a reason thatthe shoots that flowered were alive even after 4–5 months after flowering ended.Phenolic compounds contribute by regulating the oxidation of indole-3-acetic acid(IAA).Generally,depletion in phenols is an indication for the start of flower senescence.

Table 3 Comparison of biochemical properties between Bambusa nutans and Bambusa arundinacea(two-sample t test)

Cell division,elongation and differentiation are the primary cellular functions needed for organogenesis.Various studies recognized peroxidase activity as a biochemical marker to characterize the induction phase of the flowering process(Gasper et al.1985;Greppin 1986;Bouazza et al.1993).Peroxidase is a versatile enzyme involved in an array of metabolic pathways from scavenging superoxide radicals to biosynthesis of lignin for lignification of differentiating cells(Berthon et al.1989;Ansari et al.1996).Fouche and Coumans(1995)also observed an increase in peroxidase activity in leaves and internodes during flowering inVanilla planifolia.Peroxidase activity increases with stress and inactivates or destroys IAA and thus hampers plant growth(Ebrahimzadeh and Abrishamchi 2001).Lokhande et al.(2003)considered increased H2O2formation as a possible factor in flower induction inArabidopsis thaliana.However,low peroxidase activity in leaves of floweredB.nutansin the present study was associated with a long time interval between flowering and sample collection.Endogenous IAA level is directly related to flowering and its oxidation is correlated with the regulation of peroxidase activity.Flower initiation and development in bamboos is a complex morphogenic process involving metabolic changes and thus affecting the physiology of the whole culm/clump.Interaction between phenolic compounds and enzymes involved in IAA catabolism such as peroxidases might hamper vegetative growth and thus induce flowering,whereas carbohydrates provide structure and energy during the whole morphogenic process from flower induction to growth and development.

AcknowledgementsAuthors are grateful to Dr.U.Prakasham,Director and Shri P.Subramanyam,Group Co-ordinator(Research)of the Institute for providing necessary facilities and guidance and Shri Jay Prakash Mishra and Ku.Deepti Bhadrawale for technical assistance.