South Asian J Exp Biol; 10 (4): 222-233; 2020 [DOI: 10.38150/sajeb.10(4).

p222-233]

ISSN: 2230-9799 Vol. 10, Issue 4, Page 222-233 http://www.sajeb.org

REGULAR ARTICLE

Effects of Oligoarabinoxylans From Aristida pungens Leaves on

Germination and Growth of Barley
Lahouari Chaa1, Jean Claude Mollet2, Meriem Kaid-Harche1
1
Laboratoire des Productions et Valorisations Végétales et Microbiennes, Université des Sciences et de la Technologie
d’Oran Mohamed Boudiaf, USTO-MB, BP1505El M’naouar, 31000 Oran, Algerie
2
Normandie Univ, Uni Rouen, Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale (Glyco-MEV), Fédéra-
tion de Recherche "Normandie-Végétal" – FED 4277, I2C Carnot, 76000, Rouen, France
ARTICLE INFO ABSTRACT

Article History:
Received: 29 Apr 2020
Revised: 23 Jun 2020
Accepted: 27 Jun 2020
*Corresponding Author:
Email: lahouari.chaa@univ-usto.dz
Telephone: +213798808673
Keywords: Aristida pungens, oli-
goxylans, Hordeum vulgare, germi-
nation, root biomass
The objective of this study was to investigate a possible biological activities
of oligosaccharides resulting from the hydrolysis of parietal hemicelluloses of
the foliar tissue of Aristida pungens. Known for its developed, fiber-rich foliar
tissue, this perennial grasses is widely distributed on the arid and sub-
desertic regions of Algeria. In order to identify added-value application for
the cell wall hemicellulose of Aristida pungens (Poaceae), the polymers were
hydrolyzed enzymatically and after size exclusion chromatography, oligosac-
charide fragments were tested for their abilities to modify the germination
and growth of barley seeds (Hordeum vulgare). Five fractions were sepa-
rated with different compositions and molecular weights. The low molecular
weight fractions were mostly composed of oligo-xylans, while the high mole-
cular weight fractions consisted of oligo-glucuronoarabinoxylans. Biological
tests suggested that the oligosaccharide fractions and particularly the low
molecular weight fraction could promote germination and increase root bio-
mass at low concentrations or reduce it at higher concentrations, suggesting
a possible bio-stimulating application.

1. Introduction Among these polysaccharides, hemicelluloses are a

One of the characteristics of plant cells is the pre-
sence of an external envelope called cell wall. It is
in constant evolution according to the stage of
development of the tissues and environmental fac-
tors. Thus, the wall is called primary in young tis-
sues and secondary in old tissues, knowing that all
intermediate cases can be observed.
The wall is mainly composed of carbohydrate po-
lymers, including cellulose, pectins and hemicellu-
loses, proteins and ocasionnally other compounds
of a phenolic nature such as lignins (Albersheim et
al., 1977).
heterogeneous, diverses and characteristics of the
studied species. In eudicot plants, hemicelluloses
are mainly xyloglucan (Schultink et al., 2014; , De-
hors et al., 2019) whereas in monocot species, in-
cluding Poaceae, they are arabinoxylan type.
(Scheller & Ulvskov P., 2010). Arabinoxylans are
polymers composed of a D -xylopyranoses linked
in b-(1-4) backbone, which can be substituted with
L-arabinofuranose linked to the C3 of the xylose
and in some cases of 4-O-methylglucuronic acid
residues. (Scheller & Ulvskov, 2010, Bacic et al.,
1988, Harris et al., 1997).
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 Chaa et al., South Asian J Exp Biol; 10 (4): 222-233; 2020
The structural complexity of the wall indicates that
it is not only a "plant skeleton" in charge of suppor-
ting and protecting the plant, but it is also a dyna-
mic structure participating in the growth and deve-
lopment of plants (Mravec et al., 2017), in defence
processes, and is also considered a source of signal-
ling molecules (Albersheim & Darvill , 1985 ; De-
lattre et al., 2005 ; Larskaya & Gorshkova, 2015 ;
Van Aubel & Van Cutsem, 2016). Indeed, it is now
clearly accepted that oligosaccharides resulting
from the degradation of cell wall polysaccharides
can act as regulators of plant growth and develop-
ment (Laporte et al., 2007 ; Liu et al., 2010) and
induce responses to biotic and abiotic factors
(Cabrera et al., 2010 ; Rasul et al., 2012 ; Zou et al.,
2015 ; Zong et al., 2017). The biological activity of
oligosaccharide fractions depends on their degrees
of polymerization (DP), compositions and struc-
tures (Ochoa-Villarreal et al., 2012).
Some works have demonstrated that xylo-
oligosaccharides are a class of oligosaccharines,
which are bioactive oligosaccharides (Darvill et al.,
1992). These fragments consist of two to ten xylose
units (De Oliveira et al., 2018 ; Romero-Fernandez
et al., 2018), that may be substituted by other
sugar units such as arabinose and/or glucuronic
acid in α-(1-2) and in α-(1-3) (Chen et al., 2016).
Oligoxylans have been used as a substrate or addi-
tive in some bacterial productions [Chen et al.,
2016 ; Menezes et al., 2018 ; Grassi et al., 2018) or
for their antioxidant capacity (Antov & Dordevic,
2017 ; Ðordevic & Antov, 2016 ; Gowdhaman &
Ponnusami, 2015), but there has been little work
on the biological effects of this class of oligosaccha-
rides on plant growth and rooting (Ishihara et al.,
1991 ; Ishii et al., 1992 ; Katapodis et al., 2002).
For these reasons, we decided to initiate this pros-
pective study, which aims to demonstrate the pos-
sible affiliation of oligoxylans, obtained by enzyma-
tic hydrolysis of hemicelluloses extracted from the
leaf cell walls of Aristida pungens, to the oligosac-
charin family.
To our Knowledge no studies have been done on
the biological activites of hemicelluloes from leaves
of this specie which is one of the most abundant
rhizomatous grasses found in the arid and sub-arid
regions of Algeria. Aristda pungens is characterized
by the presence of a developed leaf tissue (Figure
1) (Harche et al., 1992), with an average productivi-
ty of 300-500 kg of dry matter per hectare (Djebaili,
1990) and also has a wall rich in hemicellulosic
Figure 1: Aristida pungens tuft in its naturel environment.
compounds, especially xylans (Chaa et al., 2008).
2. Material & Methods
2.1. Plant material
Hemicelluloses were extracted from leaves of Aris-
tida pungens (Poaceae, Monocot), characteristic of
Algerian sub-desert regions.
The biological effects of oligoxylans were carried
out on germination and growth of barley caryopsis
(Hordeum vulgare L. variety "colibri" Poaceae, Mo-
nocot).
2.2. Extraction of hemicelluloses
The leaves were collected in the El Kheiter region,
located in the highlands inwestern Algeria (34°
04'37.5"N 0°05'19.2"E). After harvest leaves were
dried and crushed in a knife mill.
The sequential extraction protocol was based on
methods minimizing polymer degradation
(Redgwell & Selvendran, 1986) and was adapted to
the species studied (Chaa, 2004) for the extraction
of hemicellulosic compounds.
Briefly, the plant powder was treated with a chloro-
form-methanol mixture (vol/vol) for 14 h at room
temperature with stirring. This treatment is was
repeated a second time in order to remove as ma-
ny organosoluble impurities as possible. After filtra-
tion, the residue was resuspended in 95% ethanol
for 2 h to remove traces of chloroform and then
incubated in boiling 95% ethanol for 2 h.
The insoluble residue was then dried at 50°C for 48
h. This fraction represents the alcohol insoluble
residue. From this residue, the pectic polymers
were then extracted with cold water (4°C for 2
hours), boiling water (for 2 hours) and then with
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1% ethylenediaminetetraacetic acid (EDTA) for 4 h
at 80°C. After filtration, the insoluble residue was
treated for 2 h at 80°C in a solution of NaOH (1%)
prepared in 75% ethanol to remove lignins
(Gabrielii et al., 2000).
Finally, the hemicelluloses were extracted in a 14%
KOH solution for 14 h at room temperature. The
solubilized hemicellulose fraction is was collected
from the insoluble residue by filtration. The filtrate
was neutralized with acetic acid, dialysed against
distilled water and precipitated by adding 3 vol of
95% ethanol. The polysaccharide precipitate was
recovered by centrifugation, dissolved in water and
finally freeze-dried.
2.2. Production and separation of oligoxylanes
The arabinoxylan-enriched fractions werehydro-
lysedwitha commercial recombinant endo-(1,4)-
xylanase (Sigma, X2753-10G), produced by fermen-
tation of genetically modified Aspergillus oryzae
expressing the Thermomyces lanuginosus xylanase
gene.
The enzyme mixture consisted of 500µL of xylanase
(1 unit.mL-1) and 1 mL of the xylan extract prepared
at 1% (w/v) in 50 mM acetate buffer, pH 5.4. En-
zyme incubation was carried out at 40°C for 35 min.
This duration was determined experimentally by
enzyme kinetics (0 to 70 min) and by monitoring
the progress of digestion by measuring the redu-
cing sugars with para-hydroxybenzoic acid hydro-
razyde (PAHBAH) (Fry, 1986). A xylose standard
was simultaneously carried out at 5, 10, 20, 30 and
40µg.mL-1.
At the end of the enzymatic incubation, the diges-
tion medium was placed in a boiling water bath for
5 min, then cooled and 3 vol of ethanol were added
to precipitate the undigested polymers. After cen-
trifugation at 40.000 g for 15 min at 4°C and evapo-
ration of the supernatant, the oligosaccharide ex-
tract is finally resuspended in 500 µL of distilled
water.
2.3 Separation by size exclusion chromatography
The oligosaccharide extract (500 µL) was deposited
in a low-pressure chromatographic column (1.5 ×
73 cm), filled with Biogel P2 (BioRad), connected to
a peristaltic pump with a flow rate at 0,5 mL.min-1
and a fraction collector. Fractions collected and the
elution profiles were obtained by colorimetric de-
termination of the total sugars using the phenol
with sulphuric assay (Dubois et al., 1995).
The tubes were then pooled into 5 fractions (F1 to
F5). Each of the fractions was resubmitted to a se-
cond separation on BioGel P2 by collecting 0.5 mL
per fraction and again pooled to obtain 5 fractions
(F1' to F5').
2.4. Thin layer chromatography
The collected fractions from the first and second
separation on BioGel P2 as well as the crude hydro-
lysate were deposited at 10µg, on a thin layer of
silica plate. Xylose and sucrose were used as con-
trols. The plate was placed vertically in a tank satu-
rated with a solvent mixture of butanol/acetic acid/
water (2/1/1, vol/vol/vol).
The plate was subjected to a double migration to
improve oligosaccharide separation. The sugars
were revealed by spraying the palte with the orci-
nol sulfuric reagent (49 mL water, 6 mL sulphuric
acid, 0.2 g orcinol and 145 mL 95% ethanol) fol-
lowed by incubation at 110°C for 10 min.
2.5. Liquid Gas Chromatography (GLC)
2.5.1. Preparation of samples
Fractions (100 µg) obtained after the second run on
BioGel P2 were mixed with 32.8 µg lysine, used as
an internal standard, in a hydrolysis tube and
freeze-dried.
The dry residue was subjected to a methanolysis
(methanol/anhydrous HCL 0.5 M) in a nitrogen at-
mosphere at 80°C for 20 hours. The samples were
dried under a nitrogen flow and derivatized with
heptafluorobutyrate according to the method of
Zanetta et al. (1999).
A reference mixture consisting of authentic mono-
saccharides (glucose, xylose, arabinose, galactose,
glucose, mannose, rhamnose, ribose, fucose, glucu-
ronic acid, 4-O-methyl glucuronic acid and galactu-
ronic acid) were performed in parallel to identify
the peaks and calculate the correction factors for
each monosaccharide.
2.5.2. Analysis
The derived samples were separated on a Varian
3900 type GC, equipped with a CP Sil 5-CB column
(0.5mm x 50m, fused silica, Varian) and a flame
ionization detector. The carrier gas was nitrogen
under a pressure set at 5.5 psi.
The temperature program was set at 150°C for 10
min, a temperature increase from 150 to 200°C at a
rate of 0.8°C.min-1, then a 7 min plateau at 200°C,
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a further increase from 200 to 240°C at a rate of 5°
C.min-1 and a final plateau at 240°C for 20 min. The
temperatures of the detector and the injector we-
reset at 260°C.
The sugars were identified by comparing their re-
tention times with those of the control sample and
quantified using lysine as the internal standard.
2.6. Germination Tests
2.6.1. Control calibration
In order to determine the optimal conditions for
germination of barley , several treatments and cul-
ture conditions were carried out in distilled water :
- Sterilization or not of the caryopses. The
treatment consisted of a bath of 1% sodium hypo-
chlorite for 10 min followed by 3 rinses with distil-
led water for 5 min each.
- Sealing or not of the petri dishes with parafilm
during the culture.
In all cases, germination assays were performed in
the dark in petri dishes (100 mm x 15 mm) contai-
ning two filter papers soaked with 5 mL of test so-
lution and 15 caryopses per dish.
Germination rates were determined daily by obser-
vation of the caryopses, germination being counted
positive as soon as the radicle emerged from the
integument.
2.6.2. Tests for oligosaccharide fractions
The different oligosaccharide fractions from the
second run on BioGel P2 (F1' to F5'), xylose and the
initial undigested fraction prepared in distilled were
tested at 5mg.L-1, 1mg.L-1, 100µg.L-1, 10µg.L-1,
1µg.L-1 and 0.1µg.L-1 .
The number of germinated grains was recorded
daily for the first 4 d. At the seventh day, the num-
ber and fresh mass of the roots and the length of
the coleoptiles were measured. The results were
statistically processed with StatView by comparing
the means obtained with a one-way analysis of va-
riance (ANOVA, P<0.05).
3. Results
3.1. Production and analysis of oligosaccharide
fractions
3.1.1. Separation of oligosaccharide fractions
After obtaining the Biogel P2 elution profile of the
hydrolysate, the tubes were pooled into five oligo-
saccharide fractions (F1-F5) (Figure 2A).
Each of the fractions was then resubmitted to a
second run (Figure 2B-F). Only the tubes making up
the largest part of the peak were pooled to elimi-
nate possible contamination of the peripheral frac-
tions as shown in Figure 2B-2F.
3.1.2. Separation analysis
Thin layer chromatography was carried out in order
to determine the quality of the column separation
of the different fractions and to estimate the size of
the oligosaccharide fractions (Figure 3).
The profile revealed an increasingly important mi-
gration of the oligosaccharides with the decrease of
their molecular weight. Thus, large oligosaccha-
rides (F1') did not migrate whereas the low mass
oligosaccharide fractions migrate between sucrose
and xylose as for F5'.
In addition, it is interesting to note a decrease in
the contamination after the second separation on
Biogel P2. As an example, the intensity of the con-
taminant spots (*) in F5 decreases after the second
separation in F5'. This observation also applies bet-
ween F4 and F4'.
3.1.3. Monosaccharidic analysis of fractions
The monosaccharide composition (Table I) clearly
indicated that xylose is the main monosaccharide in
all fractions. It fluctuated between 53.3% in F1' and
reached 97.7% in F5'.
Arabinose was also present in significant quantities
as it can reach more than 24% in F1'. For the other
sugars, except for glucose, which represented a
rate close to 9% in F1', they are present in rather
small quantities (less than 5%). These results con-
firm that hemicelluloses from Aristida pungens, is
of the glucuronoarabinoxylan (GAX) type.
Based on these analyses, it can be assumed that
the low molecular weight oligosaccharide fractions
(F4' and F5') are essentially oligoxylans. Larger oli-
gosaccharides are probably oligoarabinoxylans.
3.2 Germination tests
3.2.1. Control calibration
Prior to testing the oligosaccharide fractions, the
optimum condition had to be developed. For this
purpose, two conditions were carried out: one con-
sisting of a sterilization step of the caryopses be-
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Figure 2: Elution profile of the hemicellulosic hydrolysate after thefirst separation on Biogel P2 (A) and the second separa-
tion of each fraction (B-F). The fractions (F1-F5) obtained after the first run were submitted separately to a second separa-
tion. The coloured area corresponds to the tubes that were pooled to obtain the F1'-F5' fractions.

Figure 3: Thin layer chromatography of the fractions (F1-F5) obtained after the first separation of the hemicellulosic hy-
drolysate and the F1’-F’5 fractions from the second individual separation of the F1-F5. P. hemicellulosic polymer, H. crude
hemicellulosic hydrolysates (before separation), T. Standard composed of xylose (x) sucrose (s). The arrow indicates the
direction of migration.

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Figure 4: Germination and growth of a barley seedling in the presence of water.

A. Dry Grain; B. 24h ; C. 36h ; D. 48h ; E. 72h ; F. 4d ; G. 5d and H. 7d culture.
Figures 3Ha and 3Hb are enlargements of Figure 3H of the caryopsis (3Ha) and the emergence of the
coleoptile leaf (3Hb), indicated by a white arrow. Scale bar = 1cm.

Figure 5: Germination rate of barley caryopses in the presence of F5' fraction.

Figure 6: Evolution of the root fresh mass of barley seedlings grown for 7 d in the presence of F5'. The num-
ber of samples per condition > 82. The letters represent groups with significant differences at P<0.05.

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Fraction Xyl Ara Gal Glc Rha 4-0-MeGlcA Man

FHE 55.9 10.2 1.6 17.6 2.3 2.3 9.1
F1’ 53.3 24 4 8.7 5 5 -
F2’ 79 16 - 1.2 3 0.8 -
F3’ 81.5 15 - 0.5 3 - -
F4’ 96.7 1.2 - 2.1 - - -
F5’ 97.7 - - 2.3 - - -
Table 1: Monosaccharide composition (mol %) of the oligosaccharide fractions (F1'-F5').
Xyl. xylose, Ara. arabinose, Gal. galactose, Glc. glucose, Rha. rhamnose, 4-0-MeGlcA. 4-0- methyl glucuronic acid.
Man. mannnose. (-) not detected. FHE. Crude hemicellulosic fraction.

Conditions Germination rate after 3 d Number of Number and percen-

of culture (%) roots tage of seedlings
Without sterilization and without parafilm 70% ± 5 5 35 (27%)
Without sterilization and with parafilm 33% ±10 6 93 (71,5%)
With sterilization and parafilm 60% ± 9 7 2 (1,5%)
With sterilization and without parafilm 80% ± 9 Table 3: Number of roots per seedling
Table 2: Influence of a sterilization treatment of barley caryopses and their after 7 d of culture in water.
cultures in sealed petri dishes with parafilm or not on germination. n=130 seedlings
The number of dishes per condition = 2 or 30 caryopses

fore cultivation and the other by cultivating the majority of seedlings developed 6 roots.
caryopses in petri dishes sealed or not with para-
film (Table II). 3.3. Effects of the oligosaccharide fractions

The sterilization step did not seem to affect germi-
nation. However, there was an adverse effect of
sealing the petri dishes with parafilm on germina-
tion rates. The parafilm certainly reduced evapora-
tion but probably minimized gas exchanges with
the surrounding environment. Therefore, in the
following trials, the caryopses were eterilized and
grown in petri dishes not sealed with parafilm.
Under these predetermined cultural conditions, the
germination of barley caryopses followed the
pattern shown in Figure 4.
The dry caryopsis (Figure 4A) after 12-24 hours of
culture including the soaking period showed a ra-
dicle emerging from the integument (Figure 4B).
This radicle developed (Figure 4C) and several roots
appeared after 48h-72h (Figure 4D).
During this period, the coleoptile also developed to
protect the gemmule (Figure 4E-G). The first leaf
generated by the gemmule grows inside the co-
leoptile (Figure 4F-G) and pierces it (Figure 4H).
To determine whether the number of roots per
seedling could be used as a screening criterion,
trials were conducted in the presence of distilled
water and the oligosaccharide fractions (F1'-F5')
(Table III).
No significant differences were found between the
seedlings tested in each condition. In all cases, the
Three criteria were used to determine the possible
biological effects of the oligosaccharide fractions
on the germination and growth of barley seedlings:
germination rate, fresh root mass and coleoptile
length.
3.3.1. The germination rate of caryopses
The first germination kinetics (Table IV) showed
that the number of germinated caryopses increa-
sed rapidly over time. Indeed, after 48 h of culture,
almost all the grains have developed a radicle.
It appeared some effect of the oligosaccharide frac-
tions on germination rate. These effects
(acceleration or deceleration) differed according to
the fractions and concentrations studied and are
obviously more significant after 24 hours of cultiva-
tion.
Contrary to previous observations, the germination
rate of barley caryopses during the first h of culture
in the presence of F5' (Figure 5) was apparently
stimulated compared to the water control, es-
pecially at low concentrations.
The results of the Anova test showed a significant
difference at P < 0.05 of all concentrations tested
compared to the water control.
3.3.2. Root mass
The increase of the fresh root biomass of 7 day- old

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Concentration Time (in hours)

Treatment (mg.L-1) 24 48 72 96
Distilled water 71.5 89.5 96.5 100
Xylose 1 86 86 93 100
0.1 71 93 100 100
0.01 71 100 100 100
0.001 73 93 93 100
0.0001 50 93 100 100
Crude hydrolysate 1 57 100 100 100
0.1 57 93 93 100
0.01 53 100 100 100
0.001 64 93 93 100
0.0001 67 100 100 100
F1' 1 87 93 100 100
0.1 93 100 100 100
0.01 93 100 100 100
0.001 85 100 100 100
0.0001 100 100 100 100
F2' 1 33 100 100 100
0.1 86 100 100 100
0.01 80 93 100 100
0.001 87 100 100 100
0.0001 79 100 100 100
F3’ 1 60 73 80 100
0.1 62 92 92 100
0.01 53 100 100 100
0.001 60 100 100 100
0.0001 93 100 100 100
F4’ 1 86 100 100 100
0.1 73 93 100 100
0.01 69 77 100 100
0.001 71 100 100 100
0.0001 100 100 100 100
F5’ 1 29 71 79 100
0.1 47 93 100 100
0.01 71 93 93 100
0.001 53 100 100 100
0.0001 50 100 100 100
Table 4: Time course of the germination rates at 21°C of barley caryopses in the presence of increasing concentrations
of the oligosaccharide fractions (F1'- F5'), xylose, crude hemicellulosic hydrolysate or water.
n=15 (1 Petri dish per condition)

barley seedlings in the presence of F5' (Figure 6)
appeared to be dose dependent.
The F5' fraction at 0.0001 mg.L-1 significantly redu-
ced the root biomass compared to the other condi-
tions, whereas the F5' tested at 0.01 mg.L-1 had an
opposite effect. The other concentrations tested
were not significantly different from each other
and from the water control.
3.3.3. Effect on the length of the coleoptile
Measurements of the length of the coleoptiles after
7 d of culture with the same seedlings used for root
biomass measurements were also performed
(results not shown), but statistical analyses (Anova
test) did not highlighted any significant differences
between the different conditions.
4. Discussion
Enzymatic production of oligosaccharides has al-
lowed the separation of 5 more or less well sepa-
rated fractions. This separation between heavy and
light oligosaccharides was significantly improved by
the integration in the protocol of a second separa-
tion on P2 biogel.
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The quality of the enzymatic digestion was mainly
influenced by two factors, the length of the main
chain and the degree of substitution (Li et al.,
2000).
It is interesting to note that the Ara/Xyl ratio of the
oligosaccharide fractions decreases progressively
from F1' to F5'. This observation seems to indicate
a decrease in the degree of substitution of the main
xylan chain.
Therefore, it is not surprising to find low molecular
weight oligosaccharides consisting mostly of xylose
and heavy oligosaccharide populations composed
of arabinose-branched regions and 4-O-methyl glu-
curonic acid, thus rendring less accessible to xyla-
nase degradation. By studying the differences bet-
ween two endo-xylanases, isolated from Aspergil-
lus awamori, an endo I that belongs to the glyco-
side hydrolase family 10 and an endo III which be-
longs to the glycoside hydrolase family 11 (family of
enzyme, used in this study), Gruppen et al. (1993)
found that endo III was more efficient on long un-
substituted xylan fragments.
As a result, three types of oligosaccharides were
obtained, oligoxylans, oligoarabinoxylans and oligo
glucuronoarabinoxylans of varying sizes. In addition
to the glucuronoarabinoxylanic nature of the hemi-
celluloses constituting the cell wall of this Poaceae,
the detection of glucose residues in the F1' oligo-
saccharide fraction could be explained by the pre-
sence of polymer fragments of the (1,3),(1,4)-D-mix
-glucan type, characteristic of the Poaceae wall
(Buckeridge et al., 2004).
Determination of the best conditions for germina-
tion of barley caryopses allowed an increase in the
germination rates compared to preliminary trials
(temperature 20-21°C, darkness). One of the im-
portant points is to allow gas exchanges with the
surrounding environment. Indeed, the presence of
O2 is a necessary parameter for germination.
During the first trials, given the limited duration of
the work and the length of each test (7 d), the
choice to focus on one fraction out of the five pro-
ved to be crucial. From the initial observations, the
F5' seemed to have a noticeable effect, so a large
number of tests were carried out on this sample.
After 48 h of culture, the maximum germination
rate of 100% was almost reached, whatever the
concentrations used, and the germination seemed
to be faster than the control.
In relation to the effect of polysaccharides or their
derivatives on germination, the effect of chitosan
has been well documented. In 2002, Ruan and Xue
showed that covering rice seeds with chitosan
caused an acceleration of germination. Zhou et al.
(2002) obtained also an increase in the germination
percentage of peanut seeds by soaking them in chi-
tosan. In 2009, Guan et al. found that soaking
maize seeds in chitosan solution beforehand did
not have a significant effect on germination percen-
tage, but they were able to obtain a reduction in
the average germination time. Finally more re-
cently, Li et al. (2019) demonstrated an impro-
vement in germination of wheat seed following
treatment with chitosan at 50 µg.mL-1.
The effect of F5' on root biomass differed depen-
ding on the tested concentrations. When applied at
0.001 mg.L-1, F5' increased root biomass. This
effect was opposite when F5' was used at a lower
concentration (0.0001 mg.L-1). The stimulating
effect of oligosaccharides on root biomass has al-
ready been reported for oligogalactoglucomannans
in Zea mays (Kollárová et al., 2018).
In the literature, other stimulating effects of oligo-
saccharide fractions on rhizogenesis were reported,
such as the effect of chitooligosaccharides on root
biomass production in Triticum aestivum (Zhang et
al., 2016) or oligoalginates on root elongation and
induction of adventitious roots in Oryza sativa
(Zhang et al., 2014). Other oligosaccharides may
exert a double effect depending on the concentra-
tion. This is the case for oligogalacturonides which
inhibited the primary root elongation but stimu-
lated lateral root in Arabidopsis thaliana
(Hernández-Mata et al., 2010) and Zea mays (Peña-
Uribe et al., 2012).
Root development is mainly studied in relation to
the effects of different phytohormones. In 2005,
Debi et al. found a double effect of cytokinins
(kinetin and trans-zeatin) on root formation and
elongation in rice. At 1 µM, these two hormones
completely inhibited the formation of lateral roots,
while these same molecules had a stimulating
effect on lateral root elongation. The formation of
these lateral roots was also stimulated by auxin.
In 2001, Casimiro et al. found that the formation of
these roots was blocked by the auxin transport in-
hibitor N-(1-naphthyl) naphthalamic acid (NPA). In
Arabidopsis, root growth is controlled by auxin,
which acts by modulating the cellular response to
gibberellins. Xiangong and Harberd (2003) ob-
served an inhibition of rhizogenesis in Arabidopsis
230
 Chaa et al., South Asian J Exp Biol; 10 (4): 222-233; 2020
mutants, which are deficient in the production of
gibberellins, whereas in seedlings to which this
gene has been transgenically reintroduced, the
roots are the same length in wild-type seedlings.
The length of the coleoptile did not vary signifi-
cantly with the different treatments, but other con-
centrations that were not tested during this work,
as well as the other fractions (F1', F2', F3'and F4')
still need to be investigated.
5. Conclusion
Compared to the objectives fixed at the beginning
of this work, the enzymatic hydrolysis of hemicel-
luloses from Aristida pungens has allowed the se-
paration of 5 fractions with diffrent molecular
weights. These oligosaccharide fractions, and in
particular the F5' which was the most studied du-
ring this work, would seemed to have biological
effects, especially on the germination of barley ca-
ryopses. More comprehensive studies on the other
fractions are needed.
It would be equally interesting to test these oligo-
saccharide fractions on the germination of other
monocot and eudicot. The interest would be to
develop, depending on the effects, either a bioher-
bicide or a biostimulant that would be both se-
lective and natural.
Acknowledgements
The authors thank Prof. Vincent Gloaguen
(PEIRENE, Université de Limoges, France) for the
gift of 4-O-methyl glucuronic acid.
References
Albersheim P, Mcneil M, Labavitch JM (1977) Themolecular struc-
ture of the primary cell wall and elongation growth. In PiletPE (ed.)
Plant Growth Regulation. Springer-Verlag,Berlin, Heidelberg, pp.1-
12.
Albersheim P, Darvill AG (1985) Oligosaccharins. Scientific Ameri-
can 253: 85 – 64.
Antov MG, Dordevic TR (2017) Environmental-friendly technolo-
gies for the production of antioxidant xylooligosaccharides from
wheat chaf. Food Chemistry 235: 175-180.
Bacic A, Harris PJ, Stone BA (1988) Structure and function of plant
cell walls. In Preiss, J., (ed.) The Biochemistry of Plants, Academic
Press 3: 297-371.
Buckeridge MS, Rayon C, Urbanowicz B, Tiné MAS, Carpita N
(2004) Mixed linkage (1→3), (1→4)-β-D-Glucans of grasses. Cereal
Chemistry 81: 115-127.
Cabrera JC, Boland A, Cambier P, Frettinger P, Van Cutsem P (2010)
Chitosan oligosaccharides modulate the supramolecular confor-
mation and the biological activity of oligogalacturonides in Ara-
bidopsis. Glycobiology 20:775-786.
Casimiro I, Marchant A, Bhalerao RP, Beeckman T, Dhooge S,
Swarup R, Graham N, Inze D, Sandberg G, Casero P, Bennett M
(2001) Auxin transport promotes Arabidopsis lateral root initia-
tion. Plant Cell 13: 843-852.
Chaa L (2004) Etude prospective des voies de valorisation des
hémicelluloses de Poacées. Rapport D.U.E.R.A.S. Univ. Artois. 40p.
Chaa L, Joly N, Lequart V, Faugeron C, Mollet JC, Martin P, Morvan
H (2008) Isolation, characterization and valorization of hemicellu-
loses from Aristida pungens leaves as biomaterial. Carbohydrate
Polymers 74: 597-602.
Chen MH, Bowman MJ, Cotta MA, Dien BS, Iten LB, Whitehead TR,
Rausch KD, Tumbleson ME, Singh V (2016) Miscanthus x giganteus
xylooligosaccharides: Purification and fermentation. Carbohydrate
Polymers 140: 96–103.
Darvill A, Augur C, Bergmann C, Carlson RW, Cheong JJ, Eberhard S,
Hahn MG, Ló VM, Marfa V, Meyer B, Mohnen D (1992) Oligosac-
charins-oligosaccharides that regulate growth, development and
defence responses in plants. Glycobiology 2: 181-198.
Debi RB, Taketa S, Ichii M (2005) Cytokinin inhibits lateral root
initiation but stimulates lateral root elongation in rice (Oryza sti-
va). Journal of Plant Physiology 162: 507-515.
Dehors J, Mareck A, Kiefer-Meyer MC, Menu-Bouaouiche L, Lehner
A, Mollet JC (2019) Evolution of cell wall polymers in tip-growing
land plant gametophytes: composition, distribution, functional
aspects and their remodelling. Frontiers of Plant Science 10:441.
De Oliveira SM, Moreno-Perez S, Terrasan CRF, Romero-Fernandez
M, Vieira MF, Guisan JM, Rocha-Martin J (2018) Covalent immobi-
lization-stabilization of β-1, 4- endoxylanases from Trichoderma
reesei: production of xylooligosaccharides. Process Biochemistry
64: 170–176
Delattre C, Michaud P, Courtois B, Courtois J (2005) Oligosaccha-
rides engineering from plants and algae applications in biotechno-
logy and therapeutics. Minerva Biotecnologica 17:107-117.
Djebaili S (1990) Syntaxonomie des groupements préforestiers et
steppiques de l’Algérie aride. Ecologia Mediterranea 16: 231-244.
Ðordevic TR, Antov MG (2016) Wheat chaff utilization: Evaluation
of antioxidant capacity of wastestreams generated by different
pretreatments. Industrial Crops and Products 94: 649–657.
Dubois M, Gilles KA, Hamilton JK, Robers PA, Smith F (1995) Colori-
metric method fordetermination of sugars and related substances.
Analytical Chemistry 28: 350 – 356.
Fry SC (1986) Cross-linking of matrix polymers in the growing cell
walls of angiosperms. Annual Review of Plant Physiology 37: 165-
186.
Gabrielii I, Gatenholm P, Glasser WG, Jain RK, Kenne L (2000) Sepa-
ration, characterization and hydrogel-formation of hemicellulose
from aspen wood. Carbohydrate Polymers 43: 367-374.
Gowdhaman D, Ponnusami V (2015) Production and optimization
of xylooligosaccharides from corncob by Bacillus aerophilus KGJ2
xylanase and its antioxidant potential. International Journal Biolog-
ical Macromolecules 79: 595–600.
Guan YJ, Hu J, Wang XJ, Shao CX (2009) Seed priming with chitosan
improves maize germination and seedling growth in relation to
physiological changes under low temperature stress. Journal of
Zhejiang University. Science B 10 (6): 427–433.
231
 Chaa et al., South Asian J Exp Biol; 10 (4): 222-233; 2020
Grassi MCB, Carazzolle MF, Nakagawa BT, Ferrari A, Nagamatsu S,
Santos CR, Murakami MT, Pirolla RAS, Pereira GAG (2018) New
contributions for industrial n-butanol fermentation: An optimized
Clostridium strain and the use of xylooligosaccharides as a fermen-
tation additive. Biomass & Bioenergy 119: 304–313.
Gruppen H, Kormelink FJM, Voragen AGJ (1993) Enzymic degrada-
tion of water-unextractable cell wall material and arabinowylans
from wheat flower. Journal of Cereal Science 18:129-143.
Harche M, Mekhaldi A, Catesson AM (1992) Structure et architec-
ture pariétale des tissus foliaires d’Aristida pungens. Bulletin de la
société botanique de France 139, Lettres botaniques 2: 127-134.
Harris PJ, Keldman MR, Kendon MF, Mckenzie Rj (1997) Monosac-
charide compositions ofunlignified cell walls of monocotyledons in
relations to the occurrence of wall-bound ferulic acid. Biochemical
Systematics and Ecology 25:167-179.
Hernández-Mata G, Mellado-Rojas ME, Richard-Lewis A, López-
Bucio J, Beltrán- Peña E, Soriano-Bello EL (2010) Plant immunity
induced by oligogalacturonides alters root growth in a process
involving flavonoid accumulation in Arabidopsis thaliana. Journal
of Plant Growth Regulation 29: 441–454.
Ishihara M, Nagao Y, Shimizu K (1991) Physiological activity of
xylooligosaccharides. Research Report of Biomass Conversion
Program 27: 20-32.
Ishii K, Kinoshita I, Shigenaga H, Ishihara M, Shimizu K, Nishihara
H, Tanaka K (1992) The effects of oligosaccharides on tissue cul-
ture of white birch and black pine. Transactions of the Japanese
Forestry Society 103: 485-486.
Katapodis P, Kavarnou A, Kintzios S, Pistola E, Kekos D, Macris BJ,
Christakopoulos P (2002) Production of acidic xylo-
oligosaccharides by a family 10 endoxylanase from Thermoascus
aurantiacus and use as plant growth regulators. Biotechnology
Letters 24: 1413-1416.
Kollárová K, Kamenická V, Vatehová Z, Lišková D (2018) Impact of
galactoglucomannan oligosaccharides and Cd stress on maize
rootgrowth parameters, morphology, and structure. Journal of
Plant Physiology 222 : 59–66.
Laporte D, Vera J, Chandia P, Zuñiga EA, Matsuhiro B, Moenne A
(2007) Structurally unrelated algal oligosaccharides differentially
stimulate growth and defense against tobacco mosaic virus in
tobacco plants. Journal of Applied Phycology 1: 79–88.
Larskaya IA, Gorshkova TA (2015) Plant oligosaccharides – outsid-
ers among elicitors?. Biochemistry 80: 881–900.
Li K, Azadi P, Collins R, Tolan J, Kim JS, Eriksson KE (2000) Relation-
ships between activities of xylanases and xylan structures. Enzyme
and Microbiological Technology 27: 89 – 94.
Li R, He J, Xie H, Wang W, Bose SK, Sun Y, Yin H (2019) Effects of
chitosan nanoparticles on seed germination and seedling growth
of wheat (Triticum aestivum L.). International Journal of Biological
Macromolecules 126 : 91–100.
Liu H, Yang J, Du Y (2010) Synthesis of four oligosaccharides de-
rived from Paris polyphylla var. yunnanensis and their tobacco
(Nicotiana tabacum L.) growth regulatory activity. Plant Growth
Regulation 60: 69-75.
Menezes BDS, Rossi DM, Squina F, Ayub MAZ (2018) Xylooligosac-
charides production by fungi cultivations in rice husk and their
application as substrate for lactic acid bacteria growth. Biore-
source Technology Reports 2: 100–106.
Mravec J, Kračun SK, Rydahl MG, Westereng B, Pontiggia D, De
Lorenzo G, Domozych DS, Willats WG (2017) An oligogalacturonide
‐derived molecular probe demonstrates the dynamics of calcium‐
mediated pectin complexation in cell walls of tip growing struc-
tures. Plant Journal 91:534-546.
Ochoa-Villarreal M, Aispuro-Hernandez E, Vargas-Arispuro I, Mar-
tinez-Tellez MA (2012) Plant cell wall polymers: function, structure
and biological activity of theirderivatives. In: De Souza Gomes, A.
(Ed.), Polymerization. InTech, Rijeka, Croatia, pp. 63–86.
Peña-Uribe CA, García-Pineda E, Beltrán-Peña E, De La Cruz HR
(2012) Oligogalacturonides inhibit growth and induce changes in
S6 K phosphorylation in maize (Zea mays L. var. Chalqueño). Plant
Growth Regulation 67: 151–159.
Rasul S, Dubreuil - Maurizi C, Lamotte O, Koen E, Poinssot B, Al-
caraz G, Wendehenne D, Jeandroz S (2012) Nitric oxide production
mediates oligogalacturonide triggered immunity and resistance to
Botrytis cinerea in Arabidopsis thaliana. Plant Cell and Environ-
ment 35 : 1483–1499.
Redgwell RJ, Selvendran RR (1986) Structural features of cell wall
polysaccharides of onion Allium cepa. Carbohydrate Research 157:
183–199.
Romero-Fernandez M, Moreno-Perez S, De Oliveira SM, Santama-
ria RI, Guisan JM, Rocha-Martin J (2018) Preparation of a robust
immobilized biocatalyst of β-1, 4-endoxylanase by surface coating
with polymers for production of xylooligosaccharides from diffe-
rent xylan sources. New Biotechnology 44: 50–58.
Ruan SL, Xue QZ (2002) Effects of chitosan coating on seed germi-
nation and salt-tolerance of seedlings in hybrid rice (Oryza sativa
L.). Acta Agronomica Sinica 28(6): 803-808.
Scheller HV, Ulvskov P (2010) Hemicelluloses. Annual Review Plant
Biology 61:263-289
Schultink A, Liu L, Zhu L, Pauly M (2014) Structural diversity and
function of xyloglucan sidechain substituents. Plants 3:526-542.
Van Aubel G, Van Cutsem P (2016) COS-OGA stimulates plant in-
nate immunity in a cumulative process that involves salicylic acid.
Phytopathology 106: 20-21.
Xiangong F, Harberd NP (2003) Auxin promotes Arabidopsis root
growth by modulating gibberellin reponse. Nature 421: 740-743.
Zong H, Li K, Liu S, Song L, Xing R, Chen X, Li P (2017) Improvement
in cadmium tolerance of edible rape (Brassica rapa L.) with exoge-
nous application of chitooligosaccharide. Chemosphere 181: 92–
100.
Zou P, Li K, Liu S, Xing R, Qin Y, Yu H, Zhou M, Li P (2015) Effect of
chitooligosaccharides with different degrees of acetylation on
wheat seedlings under salt stress. Carbohydrate Polymers 126: 62-
69.
Zanetta JP, Timmerman P, Leroy Y (1999) Gas-
liquidchromatography of the heptafluorobutyratederivates of the
O-methyl-glycosides on capillary columns: method for the quanti-
tative determination of the monosaccharide composition of glyco-
proteins and glycolipids. Glycobiology 9: 255-266.
Zhang Y, Yin H, Zhao X, Wang W, Du Y, He A, Sun K (2014) The
promoting effects of alginate oligosaccharides on root develop-
ment in Oryza sativa L. mediated by auxin signaling. Carbohydrate
Polymers 113: 446-454.
Zhang X, Li K, Liu S, Xing R, Yu H, Chen X, Li P (2016) Size effects of
chitooligomers on the growth and photosynthetic characteristics
of wheat seedlings. Carbohydrate Polymers 138: 27–33.
232
 Chaa et al., South Asian J Exp Biol; 10 (4): 222-233; 2020
Zhou YG, Yang YD, Qi YG, Zhang ZM, Wang XJ, Hu XJ (2002) Effects
of chitosan on some physiological activity in germinating seed of
peanut. Journal of Peanut Science 31(1):22-25.

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