Effects of key growth conditions on endogenous hormone content in tillering stem bases,germination of lateral buds,and biomass allocation in Indocalamus decorus

Guibin Gao•Hao Zhong•Zhizhuang Wu•Neng Li,2•Zheke Zhong•Yanhong Pan,2•Liangru Wu,2

Abstract Physiological responses and changes in growth of Indocalamus decorus Q.H.Dai under different ecological conditions are essential for further understanding growth regulation and adaptive mechanisms and establishing an evidence-based management system for optimal growth.In this study,the endogenous hormone content in tillering stem bases,germination of lateral buds,and biomass allocation of this bamboo species in different growth environments were investigated.Among the endogenous hormones in the basal stems of tillers,indole-3-pyruvic acid and zeatin riboside were highly correlated with lateral buds that germinated to form shoots,while gibberellic acid was highly correlated with lateral buds that germinated to form rhizomes.The best lateral bud germination characteristics were achieved with full sun,a density of six plantlets per pot,and watering every 6 days.I.decorus plantlets used different resource allocation strategies depending on treatment.Different ecological factors influenced endogenous hormones in the bamboo stem base,which affected lateral bud germination and biomass allocation.

Keywords Biomass allocation·Ecological factor·Indocalamus decorus·Lateral bud ·Plantlet·Tillering

Introduction

Environmental factors play crucial roles in regulating plant growth.Physiological signals,including phytohormones and sugars,are associated with direct phenotypic changes such as bud germination(Chao et al.2007)and biomass allocation(Sugiura et al.2016;Poorter et al.2012)when plants perceive environmental signals(Chao et al.2016;Kiba et al.2011;Halliday et al.2009),to maximize their fitness(Osone and Tateno 2003;Sugiura and Tateno 2011).Because bamboo exhibits complex ecological responses in the wild(Shi et al.2014),considerable research has focused on relationships among environmental factors,gene expression,shoot development,and biomass allocation in different bamboo species(Liao et al.2013;Hishi et al.2015;Jin et al.2016;Zhuang et al.2017).These studies are relatively in-depth,but suffer from discontinuity.How the environmental factors affect endogenous hormones,initiate gene differential expression,regulate the development direction of bamboo lateral buds,and induce biomass allocation changes have rarely been studied,and this information is very important for further understanding the growth regulation and adaptation mechanisms of bamboo in response to ecological factors.

Species of bamboo can be categorized into three types based on differences in rhizome architecture:sympodial,amphipodial,and monopodial.Amphipodial bamboos have a higher sensitivity and plasticity to the external environment(Jiang 2007).Indocalamus decorus Q.H.Dai is an amphipodial species that is valued as an excellent ornamental for its beautiful appearance and clonal reproduction.It has a well-developed rhizome system that can facilitate soil and water conservation,can be widely used in landscaping and ecological restoration of abandoned mines,construction wasteland,and rocky desertified sites,and it has the potential to replace other grasses in lawns in the future.Zhuang et al.(2012,2013)analyzed the physiological response of I.decorus to elevated atmospheric ozone and CO2concentrations under the background of global climate change and found that I.decorus had good adaptability.Hu et al.(2015a,b)elucidated the physiological water integration function of I.decorus and its spacer length effects,providing a theoretical basis for irrigation of commercial bamboo forests.However,the relationships between endogenous hormone content,lateral bud germination,biomass allocation,and ecological factors in this species are not yet known.

To explore the mechanism for ecological adaptation in amphipodial bamboos to different ecological factors and thoroughly evaluate the physiological and growth response characteristics of bamboo,we examined the interactions of environmental factors with propagation of I.decorus.Potted plantlets were grown at different densities and light and watering regimes to examine the effects of these factors on endogenous hormone content,lateral bud germination,and biomass allocation to develop evidence-based strategies to manage cultivation and optimally use this valuable amphipodial bamboo.

Materials and methods

Test site and materials

Plants were grown in the open at the Taihuyuan ornamental bamboo garden in Lin′an City,Zhejiang Province,China(29°56′–30°23′N,118°51′–119°72′E).The area has a warm,humid subtropical monsoon climate with four distinct seasons.The annual precipitation is 1250–1600 mm,and the annual average temperature is 15.4°C.The monthly average temperature ranges from 3.2°C in January to 29.9°C in July,with recorded extremes of-13.3 and 40.2°C.The≥ 5°C annual accumulated temperature is 6400°C,the annual frost-free period is 235 d,and the annual sunshine hours vary between 1850 and 1950.The soil is a fertile red loam more than 60 cm deep,with a loose structure that is very suitable for growing bamboo plants.

Indocalamus decorus plantlets,which were cultivated from March 2014,were propagated from 2-year-old rhizomes from one clonal population.Mid-rhizome sections with many lateral buds were selected from one rhizome and cut into small,single rhizome section 5–6 cm long.Five to six single rhizome sections were planted in red loam soil amended with fertilizer in one pot 20 cm(diameter)by 15 cm(depth).From September to October 2014,strong,new bamboo plantlets developed from one lateral bud on each rhizome section.New shoots and rhizomes grew at the stem base of the new plantlet,and one rhizome section generally produced 3–5 new plantlets with 1–2 new rhizome(s)in a year.Rhizome sections with similarly sized plantlets were selected and pruned to remove new rhizomes and all but three plantlets.Rhizomes were replanted in rectangular pots that were 30 cm(length)by 20 cm(width)by 15 cm(depth),according to the designs outlined in the next section.Pots with two rhizomes each were established for light(60)and watering(60)treatments,while pots for density treatments(80)had variable numbers of rhizomes.All plantlets were then grown during a recovery period of 5–6 months.In February 2015,light and watering treatments were initiated.In May 2015,just when most rhizome lateral buds broke dormancy,tillering basal stem portions of plantlets were sampled to determine their hormone content.In November 2015,at the end of the annual growth cycle, final lateral bud germination of tillering stem bases and biomass of plantlets were measured.

Experimental design

Density treatments

Plants were grown in red loam soil emended with an organic fertilizer of rotten chicken manure in an 8:1 ratio(w/w).Three density treatments were established in pots:two rhizome sections,each bearing three plantlets;three rhizome sections,each bearing three plantlets;and four rhizome sections,each with three plantlets.The control had a single rhizome section bearing three plantlets.Twenty replicates were established for each treatment and for the control.Plantlets were evenly spaced within the pots and grown under full sun.The pots were watered every 6 days with a 7.5 kW pump that had a flow rate of 16.67 kg/s,and each pot received approximately 0.5 L of water each time.

Light treatments

Two light treatments were established with plastic shade nets.One layer of shade net allowed 80%light transmittance,while three layers of shade net reduced light transmittance to 40%.The control had full sun with no shading,and each treatment and the control had 20 replicates.The same soil mixture described in the previous section was used,and two rhizome sections were planted per pot.Light treatment pots received the same watering regime as density treatment pots.

Watering treatments

Two different watering frequency treatments were established,with a 6-or 9-day interval.The control was watered every 3 days.The 20 replicates for each treatment and the control received an average 0.5 L of water each time.Two rhizome sections were planted in each pot in the same soil mixture used in the density treatment,and plantlets were grown in full sun.

Methods

Endogenous hormone determination

Plantlets from the original rhizome section were primarily sampled.Five to ten pots were randomly selected from each treatment and control,and robust plantlets of a uniform size were removed from the rhizome,washed,and clipped 3–5 cm from the tillering base of the stem.Basal stem portions were weighed(fresh mass,FM)and were greater than 5.00 g.Samples were stored in dry ice during transport and endogenous hormone content was determined in the laboratory for gibberellic acid(GA),abscisic acid(ABA),indole-3-pyruvic acid(IPA),indole-3-acetic acid(IAA),and zeatin riboside(ZR)with an enzyme-linked immunosorbent assay(ELISA)(Chen et al.1998).

Lateral bud germination and biomass quantification

Ten pots were randomly selected from each treatment and control.Plant material was removed from the pots,washed,and dried.The numbers of new shoots(developed from shoot buds of tillering stem base)and rhizomes(developed from rhizome buds of tillering stem base)were counted from each original single rhizome section.For each treatment,the averages per single rhizome section and differences in lateral bud germination were compared.Rhizomes,roots,stems,and leaves were removed and weighed separately from each single rhizome section,placed in a 105°C oven for 30 min,and then dried to a constant mass at 70°C.Dry masses were recorded,and biomass per single rhizome section was compared.

Data analysis

The averages and standard deviations of stem base endogenous hormone levels,lateral bud germination number,and biomass parameters were calculated in Excel(Microsoft,Redmond,WA,USA).Correlation analysis was conducted in SPSS 17.0(IBM,Armonk,NY,USA).The treatments were compared using a one-way analysis of variance and Student–Newman–Keuls test.Unless otherwise stated,differences were significant at p＜0.05.

Results

Endogenous hormone levels,lateral bud germination,and biomass allocation of plantlets

In the density treatments(Fig.1),total hormone content(Fig.1a)was highest(4.38 ng/g FM)in stem bases from pots with two rhizome sections,followed by pots with three rhizome sections(3.96 ng/g FM),the control(3.31 ng/g FM),and pots with four rhizome sections(2.99 ng/g FM).Overall,each hormone(Fig.1a)had a significantly different level among treatments(p＜0.05).The highest numbers of shoot buds,rhizome buds,and total buds(Fig.1b)were found in the treatment with two rhizome sections,followed by the treatment with three rhizome sections,the control,and the treatment with four rhizome sections.The rhizome bud and total bud numbers were not significantly different between the treatments with two and three rhizome sections(p＞0.05),but they were significantly different from the control and the treatment with four rhizome sections(p＜0.05)(Fig.1b).The number of shoot buds was significantly lower in the treatment with four rhizome sections than in the other treatments and the control(p＜0.05)(Fig.1b).The total,rhizome,and root biomasses of I.decorus decreased with increased plantlet density(Fig.1c).Leaf and stem biomass were highest in the treatment with two rhizome sections and were significantly different from the treatment with four rhizome sections(p＜0.05)(Fig.1c).

In the light experiment(Fig.2),levels of each hormone(Fig.2a) differed significantly among treatments(p＜0.05).Levels of GA,IPA,and ZR were highest in the control,while ABA levels were the lowest(Fig.2a).Levels of IPA,IAA,and ZR were lowest in the treatment with three layers of shade net,while levels of GA and ABA were intermediate(Fig.2a).The control had the highest numbers of shoot,rhizome,and total buds,while the number of germinated lateral buds decreased gradually as shading increased(Fig.2b).The number of shoot buds and total buds was not significantly different between the control and the treatment with one layer of shade net(p＞0.05),but both were significantly different from the treatment with three layers of shade net(p＜0.05)(Fig.2b).The number of rhizome buds was significantly different between the control and the shading treatments(p＜0.05),but there were no significant differences between shading treatments(p＞0.05)(Fig.2b).The totaland individual component biomasses decreased with increased shading(Fig.2c).There were significant differences among total,rhizome,and root biomass in each treatment(p＜0.05)(Fig.2c).There was a significant difference in stem biomass between the control and the shading treatments(p＜0.05),but there were no significant differences between shading treatments (p＞0.05)(Fig.2c);leaf biomass did not vary significantly(p＞0.05)(Fig.2c).

In the watering experiment(Fig.3),GA,IAA,and ZR levels were highest in the treatment with a 6-day interval,but the ABA level was the lowest and was significantly different from the other treatment and the control(p＜0.05)(Fig.3a).The control had a relatively higher ABA content,but had the lowest GA,IPA,and IAA contents(Fig.3a).The treatment with a 6-day interval had the most germinated lateral buds,followed by the 9-day interval treatment and the control(Fig.3b).The number of germinated lateral buds was significantly different between the treatments and the control(p＜0.05),but no significant differences were found between the treatments(p＞0.05)(Fig.3b).There were no significant differences amongtotal biomass and stem biomass of each treatment(p＞0.05)(Fig.3c),but rhizome and root biomasses were significantly lower in the control than in the treatments(p＜0.05),and the control had a significantly higher leaf biomass(p＜0.05).

Fig.2 Effect of light treatments on endogenous hormone levels(a),lateral bud germination quantity(b)and biomass allocation(c).From a to c,the significance of the difference gradually strengthened(p＜5%)

Correlations between endogenous hormone levels,lateral bud germination,and biomass allocation

In the density treatments(Table 1),stem base levels of ABA,IPA,and ZR in I.decorus had significant or very significant positive correlations with the number of lateral buds that germinated(p＜0.05).The number of germinated shoot buds was more highly correlated with thesethree hormones than with the others.Lateral bud germination was highly correlated with aboveground biomass but was weakly correlated with underground and total biomass(Table 1).

Fig.3 Effect of watering treatments on endogenous hormone levels(a),lateral bud germination quantity(b)and biomass allocation(c).From a to c,the significance of the difference gradually strengthened(p＜5%)

In the light experiment(Table 1),there were significant or very significant positive correlations between stem base IPA and ZR levels and germinated shoot buds(p＜0.05).IAA had the highest correlation with germinated shoot buds while GA had the highest correlation with germinated rhizome buds,but neither had significant differences between treatments(p＞0.05).Lateral bud germination had a significant or very significant positive correlation with total biomass(p＜0.05)(Table 1)and had a higher correlation with underground biomass than with aboveground biomass(Table 1).

In the watering experiment(Table 1),there were very significant positive correlations between levels of stem base GA and IAA and lateral bud germination(p＜0.01).Correlations between germinated lateral buds and rhizome,stem,and total biomass were all very significant(p＜0.01)(Table 1).Lateral bud germination was positively but not significantly correlated with root biomass(p＞0.05),and it was negatively correlated with leaf biomass.

Table 1 Correlations between endogenous hormone levels,lateral bud germination quantity and biomass allocation in the density,light and water treatments

Discussion

As signal molecules in plants,hormones have very important functions in regulating various developmental processes and environmental responses(Schmelz et al.2003;Santner et al.2009).In general,hormones bind to receptors and initiate downstream gene expression,leading(through signal transduction)to relevant physiological or biochemical responses that influence the morphogenesis of new organs(Tian et al.2008;Torrigiani et al.2012).In the present study,hormone levels among different treatments varied greatly,and a strong correlation between hormone changes and lateral bud germination was found.Overall,GA was primarily correlated with rhizome bud germination,while IPA and ZR were primarily correlated with shoot bud germination.These results are consistent with the findings of Bowman and Floyd(2008)that low concentrations of hormones are sufficient to regulate bud growth and development.Plant hormones have key functions in regulating branching patterns and directly influence the survival and competitive abilities of plants(Assuero and Tognetti 2010;Wang and Li 2011).Differences in ecological factors resulted in different levels of endogenous hormones in the tillering stem bases of I.decorus,which affected regulation of branching development and led to differences in the production of new shoots and rhizomes.

A plant coordinates the growth of different organs as it develops.This coordinated development is affected by endogenous factors(De Kroon et al.2005;Walter and Schurr 2005),but more importantly,it is also directly or indirectly influenced by the specific environment(Kerkhoff and Enquist 2006;Violle et al.2007;Jung et al.2010).The present study noted that high and low bamboo plantlet densities were not conducive to lateral bud germination.The number of germinated lateral buds was highest under full sun,it decreased as the amount of shading increased,and it was best with the 6-day watering interval.Differences in density,light,and watering regime all affected lateral bud germination.Lateral buds provide the starting point for bamboo modular growth and development.In amphipodial and monopodial bamboo species,lateral buds on the rhizome can germinate and grow into new rhizomes or new shoots,so lateral bud differentiation determines the formation of different modules during clonal growth.Therefore,differences in lateral bud germination that are caused by different ecological factors lead to changes in bamboo morphology and physiology.

Resource allocation patterns of plants in different environments reflect the developmental allocation strategies that are determined by the environment(Kerkhoff and Enquist 2006;Violle et al.2007;Jung et al.2010).In this study,different treatments induced differences in biomass allocation,which were highly correlated with lateral bud germination.Thus,the effects of ecological factors on biomass allocation were achieved indirectly by regulating the lateral bud germination,which reflect the complex ecological adaptations of plants to environmental changes.Furthermore,it was observed that the highest average root biomass allocation(32%of the total biomass)was seen in the density experiment,with decreasing biomass allocation to rhizomes,leaves,and stems.In the light experiment,the highest average biomass allocation was to leaves,and biomass of all parts decreased as the degree of shading increased.In the watering experiment,the average root biomass allocation was highest and accounted for 30%of the total biomass,as reported in other studies that found that when water and nutrients were scarce,plants allocated more biomass to their root systems to acquire more water and nutrient resources,while in light-limited environments,plants allocated more biomass to aboveground parts to increase light acquisition(Müller et al.2000;King 2003;Coyle et al.2008).The optimal allocation hypothesis of plant resources states that plants respond to the environment primarily by regulating biomass allocations of each module to maximize gains in light,nutrients,and water resources(Bloom et al.1985;Gedroc et al.1996;McConnaughay and Coleman 1999).As organs and physiological functions respond,plants maximize their use of restricted resources to maintain their growth and reproduction(Shipley and Meziane 2002;Poorter et al.2012).The results of the present study supported the hypothesis to a certain extent.

Conclusions

In this study,treatments with different ecological factors changed endogenous hormone levels in tillering stem bases of I.decorus.GA primarily promoted rhizome bud germination,and IPA and ZR mainly promoted shoot bud germination,suggesting that hormonal changes in fluenced bud differentiation(rhizome vs shoot).Lateral bud germination can be regulated via exogenous hormones when propagating and cultivating this species.Lateral buds germinated best under full sun and at suitable plantlet densities and watering intervals,indicating that I.decorus should be grown in the open at a moderate density with sufficient moisture during cultivation.Variations in ecological factors affected lateral bud germination,induced changes in biomass accumulation and allocation,and led to differences in growth and the vegetative reproduction capacity of the clones.