Introduction
During their life-span, land plants, being sessile, have
to face sporadic climatic perturbations (Becklin et al. 2016). Light is a factor of key
importance as almost all the biological phenomena in plant are directly or
indirectly rely upon light. A mild to severe decline in the available light is
amongst the critical limiting factors for photosynthesis and plant growth.
Under sub-due light the plants dramatically change their morphogenetic pattern
by portraying etiolation (Svriz
et al. 2014; Tiryaki
and Kaplan 2019). The etiolated plants show long hypocotyls, week cell walls
and extensive loss of photosynthetic pigments, especially the chlorophylls with
the conversion of chloroplasts to etioplasts (Liu et al. 2017). This underlines that
etiolation is a sub-optimal condition, arising from low- or non-availability of
light, initiating an array of molecular and physiological changes and leading
eventually to a ceased pigment synthesis and weakening of the cell wall of
internodes (Sinclair et al. 2017).
The PGRs
are of pivotal importance in triggering key processes in plants (Symons and
Reid 2003). In agricultural practices, the growth promoters and retarders are
used to improve crop productivity when applied via different modes. The
exogenous application of PGRs is effective in enhancing the crop growth and
productivity under optimal and sub-optimal conditions. Among five naturally
occurring PGRs, gibberellins mobilize the seed reserves during germination,
promote elongation growth of stem, and trigger transition from vegetative to
reproductive growth (Binenbaum et al. 2018). Auxins signal the plant processes like cell
expansion, root branching and fruit development (Petrasek
et al. 2019). The cytokinins
promote the cell division in the meristematic tissues, delay senescence, and
promote chlorophyll synthesis (Klíčová et al. 2004; Petrasek
et al. 2019). Abscisic acid closes
the stomata, induces seed and bud dormancy and hastens senescence (Tardieu et al. 2010). Gaseous hormone ethylene
controls the ripening of climacteric fruits, floral induction, plant sex
determination and promotion of abscission (Klíčová
et al. 2004; Kabir
et al. 2018). In addition, there are
some recently introduced PGRs and chemical substances, which modulate an array
of plant processes. For instance, brassinosteroids
control cell elongation, gravitropism, maintain apical meristem and root hair
differentiation (Wei and Li 2016; Peres et
al. 2019). Jasmonates play roles in wound
response and herbivory resistance (Katsir et al.
2008; Koo and Howe 2009). Similarly, strigolactone
promote the seminal and adventitious root formation (Sun et al. 2016). At the same time, triacantonol
promotes photosynthesis, proteins synthesis, water and nutrient uptake (Naeem et al.
2012; Sharma et al. 2018).
One of the
main aspects of light and growth regulators in plants is the elicitation of
morphogenetic responses with the activation of the photoreceptors and their
interacting factors (e.g., phytochrome interacting factors; PIFs) during
seed germination and seedling growth (Lau and Deng, 2010; Pham et al. 2018). Studies show that there is
interplay between light and hormone signaling pathways for the chlorophyll
biosynthesis during the etiolation-de-etiolation transitions (Liu et al. 2017). The proteins such as PIFs, ELONGATED HYPOCOTYL 5,
ETHYLENE INSENSITIVE 3 and DELLA are key transcriptional regulators
in light and hormonal signaling pathways (Liu et al. 2017). Using altered
meristem program (Amp) 1 mutant
of Arabidopsis it has been revealed that cytokinin or cytokinin-mediated
processes are the regulators of etiolation response by acting as a component of
the induction of morphogenetic processes via signal transduction pathway, which
may be independent of light (ChinAtkins et al. 1996). During the search for
mutants showing a synergistic hormonal stimulatory response, a novel allele 7
of amp1 (amp1-7) mutant was found to show coactive stimulatory effect of
ethylene and gibberellins (Saibo et al. 2007).
It is
evident from the above that both light and PGRs intimately interact with each
other in modifying the plant morphogenetic responses during seed germination
and seedling growth. While the exogenous application of PGRs can partially
replace the requirement of light during seed germination (Sawada et al. 2008; Miransari
and Smith 2014), there is no report of the hormonal regulation in etiolated
seedlings. To test the hypothesis that etiolation is a modifying factor and
that the foliar spray of PGRs may mitigate the negative effects of etiolation
by producing profound changes, this study was performed to explore the possible
bio-regulatory role of foliar spray of optimized levels of selected PGRs
including ascorbic acid (AsA), thiourea (TU), cycocel (CCC) and kinetin (Kin) in the non-etiolated,
etiolated and de-etiolated maize seedlings based on an array of growth and
physiological attributes.
Materials and Methods
Plant
material and growth conditions
Maize (Zea mays L. Hybrid
P-1543) caryopses were obtained from Poineer Seed
Co., Sahiwal, Pakistan. The experiments were conducted in the growth room facility
of the Department of Botany, University of Agriculture, Faisalabad,
Pakistan. Ten seeds were sown in plastic pots (20 cm long, 40 and 30 cm
diameter at the top and bottom, respectively). Each pot contained 2 kg of the
washed sand. The caryopses were surface sterilized with 35% H2O2
for 15 min followed by thrice rinsing with sterilized water, and sown after
adding water in the pots (60–70% moisture). Three days after germination, the
seedlings were supplemented with 250 mL per pot of half-strength nutrient
solution (Hoagland and Arnon 1950). The uniform sized
seedlings were grown for six days in each pot at 350–400 µmol/m2/s
of while light (supplemented with LEDs), 60% RH and day/night temperature of
27±2oC/23±2oC and 14/10 h day/night.
Selection
and optimization of the pgr levels
In this study we used four PGRs with contrasting modes
of action but all these are used to improve the growth and yield of field
crops. Among the foliar-sprayed PGRs, AsA acts as
cofactor in the activities of different enzymes, triggers phytochrome mediated
signaling (Barth et al. 2006; Farooq et al.
2013), regulates cell cycle and cell elongation (Gallie
2013; Ivanov 2014) and augments plant defense (Lu et al. 2019). TU, having different
functional groups, exhibits growth bioregulatory effects in a number of plants
both under stress and non-stress conditions (Wahid et al. 2017). The CCC is a synthetic growth retardant and shortens
the intermodal length by reducing cell division and cell elongation, and
diverting assimilates for increasing grain yield (North et al. 2010; Kumar and Sharma 2019). Kin improves growth and gas
exchange properties (Shah 2007) and enhances chlorophyll synthesis by
stimulating the 5-aminolevulinic acid synthesis (Yaronskaya
et al. 2007).
In order to
optimize the most effective levels of these PGRs, range of ascorbic acid (AsA; 0–1.25 mM), thiourea (TU; 0–25 mM), cycocel (CCC; 0–2 mM) and kinetin (KIN; 0–20 mM)
levels was foliar sprayed on three days old seedlings and these seedlings were
grown for six days. In addition to an unsprayed set of plants, another set of
plants foliar sprayed with distilled water was run alongside as positive
control. At harvest the data were recorded for seedling length, dry weight and
seedling length/dry weight ratio. Optimal levels of AsA,
TU, CCC and KIN were 0.5, 10, 1.2 and 3 mM, respectively, which were
used for foliar application on non-etiolated, etiolating and de-etiolating
maize seedlings.
Etiolation
and de-etiolation treatments
Three sets of maize seedlings were grown in the pots to
find out growth and physiological changes in the non-etiolated, etiolated and
de-etiolated maize seedlings. For etiolation, before shifting to darkness, the
seedlings were foliar sprayed with distilled water and pre-optimized levels of
the selected PGRs. These seedlings were grown for six days in the darkness and
then measured for growth and physiological attributes. Another set of pots was
running in parallel in the darkness for etiolation. These six days etiolated
seedlings were foliar sprayed with the above mentioned levels of PGRs and kept
in the light for de-etiolation under the above mentioned growth conditions for
six days. A non-etiolated set of plants but foliar sprayed with water and
PGRs for six days was run alongside for comparison. In all, there were 18
treatments; each replicated thrice with 10 uniform seedlings per replication.
Experimental design was completely randomized with etiolation conditions and
hormones as two factors.
Harvesting
After six days, non-etiolated, etiolated and
de-etiolated maize seedlings (age 18 days) were harvested and measured for
different growth and physiological attributes. The harvested seedlings were
transferred to zip-lock bags, instantly frozen, and stored in a deep freezer
(Sanyo, Japan) at -40oC until used.
Growth,
pigments and gas exchange measurements
At harvest, the seedlings were measured for length, dry
weight and length to dry weight ratio. Shortly after harvest, seedlings were
chopped, ground in coarse sand using 80% acetone, filtered and absorbance of
the extract was taken at 490, 645 and 663 nm using spectrophotometer (Model
UV-1100, Shanghai, China). The contents of Chl a and
b were calculated by the method of Arnon (1949) while those of carotenoids (Car) was measured with Davies (1976)
method. Total chlorophyll (Chl) contents were calculated by summing up Chl a and b,
while Chl a/b ratio was also computed.
On the 6th
day, the leaf gas exchange parameters were measured. Fully expanded leaf from
the top was selected to determine net rate of photosynthesis (Pn),
transpiration rate (E), stomatal
conductance (gs)
and sub-stomatal CO2 concentration (Ci) using
Infra-red Gas Analyzer (IRGA; Model LCA 4, Analytical Development Co. Ltd.,
Hoddesdon, England) equipped with narrow leaf chamber. The set of conditions
for these determinations was: molar air flow per unit leaf 331 mM/m/s,
atmospheric pressure 99.8 kPa, photosynthetically active radiations on leaf
surface 374 µmol/m2/s, CO2 concentration 388 µmol/mol and ambient temperature 25±2oC.
All measurements were made in triplicate.
Oxidative
stress parameters
Both H2O2 and
For the measurement
of
MDA (nmol/g
fresh weight) = [(A532-A600)/1550000] × 106.
Antioxidants
The analysis was done for soluble phenolics (PHE)
flavonoids (FLA), anthocyanins (ANT), ascorbic acid (AsA),
niacin (NIA) and riboflavin (RIB). For the estimation of PHE contents, with the
method of Julkunen-Tiitto (1985), fresh plant
material was extracted in 80% acetone in a water bath at 50oC for 1
h. After centrifugation at 12000 × g,
100 µL of the supernatant was diluted with d.H2O, mixed and
added 0.5 mL of the Folin Phenol reagent and vortexed
for 5 sec. The 2.5 mL of 20% Na2CO3 was added and
vortexed for 5 sec, waited for 20 min and absorbance taken at 750 nm; 80%
acetone was used as blank. For the estimation of FLA contents, 1 mL of the
above acetonic extract was mixed with 4 mL water in measuring flask, mixed and
added 0.6 mL of NaNO2 and 0.5 of 10% AlCl3 after 5 min
and 2 mL of NaOH after 1 min. After adding 2.4 mL of d.H2O, the
reaction mixture was shaken well, let it stand at room temperature and measured
the absorbance at 510 nm, using 80% acetone as a blank (Zhishen et al. 1999). For the quantification of
ANT, 0.1 g of fresh material was extracted with 1 mL of acidified methanol (1%
v/v HCl) at 50oC in a water bath for 1 h and filtered. The
absorbance of the filtrate was taken at 535 nm using acidified methanol as
blank (Stark and Wray 1989).
To quantify
the contents AsA in the plant samples, 0.25 g of
fresh material was ground in 6% TCA and filtered. One mL of the filtrate was
mixed with 1 mL of dinitrophenyl hydrazine and 1 drop of 10% TU (prepared in
70% ethanol). Boiled the mixture for 10 min, cooled in an ice bath, warmed to room
temperature and added 1 mL of 80% H2SO4. The absorbance
of the solution was taken at 530 nm using TCA as blank (Mukherjee and Choudhuri 1983). To quantify NIA with the method of Okwu and Josiah (2006), 0.1 g of the fresh sample was
treated with 1 mL of 1 N H2SO4 for 20 min. After adding a
drop of ammonia, the solution was filtered and 1 mL of filtrate was mixed with
0.5 mL of 10% of potassium cyanide solution followed by the addition of 0.5 mL
of 0.02 N H2SO4. The reaction mixture was shaken well,
let stand at room temperature and absorbance noted at 470 nm. To measure the
RIB, 0.5 g of the fresh material was ground with 50% ethanol and filtered.
Filtrate (1 mL) was mixed with 5% KMnO4 solution and 1 mL of 30% H2O2,
and heated the mixture at 50oC for 30 min. After cooling, 0.2 mL of
Na2SO4 solution was added and diluted the reaction
mixture to 5 mL. After 5 min the upper colorless
layer was aspirated and its absorbance was measured at 510 nm. Ethanol (50%)
was used as blank (Okwu and Josiah 2006).
Statistical
analysis
Ten seedlings were taken per replication for the
measurement of different growth and physiological attributes. Two-way analysis
of variance (ANOVA) and comparison of treatment means (P = 0.05) were performed and treatment means were computed by
applying LSD test using STATISTIX.8.1 software. Trend-lines and correlations
were drawn using MS Excel (v. 2010) to find out any possible association of
shoot dry weight and different physiological parameters separately for
non-etiolated, etiolated and de-etiolated treatments.
%20801-810_files/image002.png)
Fig. 1: Changes in growth traits of non-etiolated (Non-Et),
etiolated (Et) and de-etiolated (De-Et) maize
seedlings with or without the foliar spray of water and PGRs. *, P < 0.05); ns, P > 0.05. Means sharing the same letter differ non-significantly (P > 0.05)
Results
Seedling
growth data
With non-significant (P > 0.05) difference among the treatments in non-etiolated
seedlings, the KIN spray was the most effective (16%) in enhancing the shoot
length followed by TU (15%) while CCC declined it (16%). Etiolation for six
days let the seedlings to elongate and a maximum elongation was noted in water
sprayed seedlings while a minimal elongation was noted with CCC spray (28%).
Foliar spray of PGRs during seedling de-etiolation nearly ceased the seedlings
elongation with the greatest decline in shoot length was achieved with CCC
(28%) followed by KIN (12%) while water spray enhanced this attribute by 2%
(Fig. 1). With significant difference in the treatments (P < 0.01), seedling dry weight data revealed that under
non-etiolation all foliar spray treatments significantly enhanced shoot dry
weight but the increase was maximum with CCC (32%) and KIN (24%) spray. Under
etiolation the seedling dry weight did not differ much with foliar spray of PGRs.
However, under de-etiolation, AsA and CCC were the
most effective in enhancing the seedling dry weight (18% each) among all the
treatments (Fig. 1). Showing significant (P
< 0.01) difference among treatments, shoot length-to-dry weight ratio was
legibly reduced with CCC spray (36% of control) followed by water spray (12%)
in non-etiolated seedlings. In etiolated seedlings, this ratio was reduced
highly with CCC (28%) and KIN (13%) spray. Under de-etiolation, shoot
length-to-dry weight ratio was the lowest with CCC spray (37%) followed by KIN
spray (19%) among all the foliar spray treatments (Fig. 1).
%20801-810_files/image004.png)
Fig. 2: Changes in photosynthetic pigment contents of
non-etiolated (Non-et), etiolated (Et) and
de-etiolated (Det) maize seedlings with or without
the foliar spray of water and PGRs. *, P
< 0.05); **, P < 0.01. Means
sharing the same alphabet differ non-significantly (P > 0.05)
Photosynthetic
pigments
Although all the foliar spray treatments increased Chl a contents in non-etiolated plants, KIN
was the most effective (14%) followed by AsA (13%).
Etiolated seedlings indicated a considerable loss of Chl a while CCC and KIN were more promising in reducing the
etiolation-induced loss of Chl a. In
de-etiolated seedlings, however and greater de
novo synthesis of Chl a was recorded with PGRs spray; KIN (44%) and CCC
(42%) were more effective than the other PGRs (Fig. 2). KIN (40%) followed by AsA (34%) were more effective in improving the Chl b contents in non-etiolated seedlings.
The Chl b content decline drastically in
etiolated seedlings and a greatest decline (39%) was noticed in water sprayed
seedlings but the least (11%) in TU treated leaves. De-etiolation recouped the Chl b content with all the PGR treatments
whereas KIN (16%) was the more effective among the PGRs (Fig. 2). In non-etiolated
seedlings, Chl a/b
ratio was reduced with all the foliar spray treatments. The etiolation declined
this ratio irrespective of the PGRs foliar spray but a maximum decline (22%)
was noted with TU spray (Fig. 2). Contrarily, in de-etiolated seedlings, there
was a substantial regain in the Chl a/b ratio; being greater with CCC (29%)
followed by KIN (26%) spray (Fig. 2). The Car
contents in non-etiolated seedlings increased with all the foliar spray
treatments while the KIN with 12% increase was the most effective. Under
etiolation treatment, the Car
contents declined markedly in all the treatments although foliar spray with CCC
and AsA was the most effective (increased by 26 and
21%, respectively) showing a lesser decline in this attribute. However, there
was a substantial increase in Car
contents of de-etiolated seedlings in all the foliar spray treatments;
nonetheless, the foliar spray of CCC was the most effective (38%) followed by
KIN (35%) in improving this pigment (Fig. 2).
Gas
exchange characteristics
In non-etiolated seedlings, all the PGRs spray increased
Pn; CCC
(25%) followed by AsA (23%) were more effective.
Under etiolation, although there was 62% decline in Pn than in non-sprayed plants,
the foliar spray of KIN and TU reduced this decline (44% each). Under
de-etiolation, there was a gain in Pn approaching nearly the level of non-etiolated plants
(Table 1). All the foliar spray treatments under non-etiolation improved E; being the highest with KIN (23%)
followed by CCC (18%) spray. In etiolated seedlings, E declined tangibly although TU spray was more effective amongst
all PGRs. De-etiolated seedlings displayed a gain in E with PGRs spray approaching the non-etiolated plants while KIN
(with 20% increase) was highly effective (Table 1). Although all the foliar
spray treatments increased gs as compared to non-etiolated seedlings, TU and
AsA (with 12 & 11% increase, respectively) were
more effective. Etiolation of plants for six days, revealed a substantial
decline in gs
although foliar spray of AsA (20%) and KIN (17%) was
highly effective. In de-etiolated seedlings, gs was recouped with
all foliar spray treatment but KIN (22%) and CCC (20%) were more effective
(Table 1). The Ci remained consistent
across all the foliar spray treatment in non-etiolated seedlings. Etiolated
seedlings indicated a large increase in this attribute, but a lower increase was
produced by KIN (29%) and TU (28%). In de-etiolated seedlings, there was a
noticeable decline in this attribute; while among the foliar spray treatments
CCC (14% decline) and KIN (13% decline) were more effective (Table 1).
Oxidative
damage parameters
In non-etiolated seedlings, the foliar spray of PGRs
declined the H2O2 contents in maize plants with a highest
decline with CCC (18%) and KIN (17%). Etiolated seedlings indicated enormously
increased tissue H2O2 level although a least accumulation
was measured in KIN (34%) and TU (33%) sprayed seedlings. In de-etiolated
seedlings there was an ample decline in H2O2 contents but
a highest decline (32%) was noted in KIN sprayed seedlings (Fig. 3). Under non-etiolated
condition MDA content was reduced by ~11% with foliar spray of PGRs. Etiolated
seedlings showed ~3-folds higher MDA which was distinctively reduced with PGRs
spray. However, in de-etiolated seedlings, the foliar spray of PGRs declined
MDA but a higher decline was noticed in KIN (40%) and AsA (38%)
sprayed seedlings (Fig. 3).
Table 1: Some gas exchange characteristics of control
(non-etiolated), etiolated and de-etiolated maize seedlings with or without the
foliar spray of PGRs
|
Etiolation (Et) treatments |
Foliar spray
(FS) |
Net rate of photosynthesis (µmol/m2/s) |
Transpiration rate (mmol/m2/s) |
Stomatal conductance (mol/m2/s) |
Sub-stomatal CO2
level (µmol/mol) |
|
Non-etiolated |
No spray |
18.61±1.24ef |
3.65±0.21d-f |
0.337±0.014c |
239.7±16.1 |
|
Water |
19.64±1.42de |
3.67±0.26c-f |
0.344±0.016bc |
240.9±21.8 |
|
|
AsA |
22.95±1.38ab |
4.10±0.21a-d |
0.375±0.015a |
231.1±16.1 |
|
|
TU |
21.09±1.52b-d |
3.99±0.24b-d |
0.378±0.017a |
241.2±19.8 |
|
|
CCC |
23.35±1.33a |
4.30±0.24ab |
0.346±0.017bc |
238.5±17.8 |
|
|
Kin |
22.65±1.37a-c |
4.49±0.33a |
0.368±0.023ab |
230.2±17.2 |
|
|
No-et→Et |
No spray |
7.14±0.76i |
2.65±0.26i |
0.245±0.017fg |
353.4±18.1 |
|
Water |
8.75±0.86hi |
3.32±0.21e-h |
0.225±0.015g |
344.7±14.4 |
|
|
AsA |
9.48±0.75h |
2.96±0.22h-i |
0.294±0.013de |
314.8±20.0 |
|
|
TU |
11.71±1.07g |
3.26±0.22f-h |
0.268±0.015ef |
309.5±13.0 |
|
|
CCC |
12.61±1.30g |
3.11±0.18gh |
0.270±0.014ef |
315.7±15.9 |
|
|
Kin |
12.58±1.14g |
3.11±0.29gh |
0.286±0.020e |
297.8±15.9 |
|
|
Et→Det |
No spray |
17.36±1.43f |
3.42±0.26e-g |
0.321±0.018cd |
269.9±10.1 |
|
Water |
16.85±1.07f |
3.24±0.28f-h |
0.328±0.019c |
268.8±20.0 |
|
|
AsA |
20.58±1.90c-e |
4.04±0.38a-d |
0.369±0.014ab |
238.9±11.3 |
|
|
TU |
20.78±1.56cd |
3.72±0.43c-e |
0.347±0.012bc |
243.0±11.3 |
|
|
CCC |
21.09±1.43b-d |
4.06±0.27a-d |
0.385±0.016a |
233.0±15.8 |
|
|
Kin |
21.15±1.16b-d |
4.11±0.32a-c |
0.393±0.018a |
236.0±19.9 |
|
|
|
SE (Et × FS) |
1.056* |
0.224* |
0.013* |
ns |
*, P < 0.05; **, P < 0.01; ns, P >
0.05. Means sharing same alphabet differ non-significantly (P > 0.05)
Phenolics
accumulation
In non-etiolated seedlings foliar spray of the PGRs
enhanced the content of PHE; being the highest due to CCC spray (27%).
Etiolated seedlings exhibited 35–50% reduction in PHE in non-sprayed and
water-sprayed seedlings but foliar spray of the PGRs was quite effective in
curtailing the loss of PHE; the loss was the lowest (26%) with AsA spray. In the de-etiolated seedlings, there was a
regain in the PHE contents with spray of all the PGRs but CCC with 32% regain
was the most promising (Table 2). The PGRs spray on non-etiolated seedlings
indicated up to 19% (with CCC) accumulation of FLA. Non-sprayed etiolated
seedlings suffered a large decline in FLA contents but foliar spray of growth
regulators incurred a lesser decline; being the lowest (30%) with KIN spray.
However, spray of the PGRs on de-etiolated seedlings recuperated the decline in
FLA contents and maximum increase (22%) was achieved with foliar spray of KIN
(Table 2). Foliar spray of PGRs on non-etiolated seedlings improved the ANT
content, and a greatest increase (17%) was recorded with CCC spray. Etiolated
seedlings indicated 41–50% decline in ANT content but the lowest loss in this
metabolite was observed with AsA spray. In
de-etiolated seedlings the PGRs spray legibly improved the ANT content, but CCC
was the most effective (Table 2).
%20801-810_files/image006.png)
Fig. 3: Changes in the oxidative stress parameters of
non-etiolated (Non-Et), etiolated (Et) and
de-etiolated (De-Et) maize seedlings with or without the foliar spray of water
and PGRs. **, P<0.01. Means
sharing the same alphabet differ non-significantly (P>0.05)
Vitamins
accumulation
Table 2: Accumulation of phenolic compounds and some vitamins in
control, etiolated and de-etiolated maize seedlings with or without the foliar
spray of water and PGRs
|
Etiolation (Et) treatments |
Foliar spray
(FS) |
Soluble phenolics (µg/g
fresh weight) |
Flavonoids (µg/g fresh weight) |
Anthocyanins (A535) |
Ascorbic acid (µmol/g fresh
weight) |
Niacin (µmol/g fresh weight) |
Riboflavin (µmol/g fresh
weight) |
|
Non-etiolated |
No spray |
30.30±1.83de |
7.39±0.45 |
0.350±0.015b |
6.24±0.51 |
0.64±0.04 |
5.04±0.30 |
|
Water |
31.62±1.83cd |
8.07±0.58 |
0.358±0.018b |
6.65±0.45 |
0.68±0.04 |
5.41±0.38 |
|
|
AsA |
35.05±1.96b |
8.32±0.47 |
0.406±0.025a |
6.94±0.42 |
0.69±0.04 |
5.61±0.31 |
|
|
TU |
33.85±2.26bc |
8.12±0.54 |
0.397±0.012a |
6.78±0.45 |
0.68±0.05 |
5.56±0.36 |
|
|
CCC |
38.49±1.46a |
8.81±0.55 |
0.410±0.031b |
6.49±0.44 |
0.68±0.05 |
5.76±0.36 |
|
|
Kin |
35.55±1.60ab |
8.23±0.51 |
0.397±0.017b |
6.79±0.49 |
0.68±0.05 |
5.80±0.50 |
|
|
No-et→Et |
No spray |
17.66±1.92g |
4.38±0.38 |
0.192±0.006f |
2.43±0.13 |
0.31±0.01 |
2.83±0.31 |
|
Water |
17.65±1.00g |
4.81±0.28 |
0.193±0.013f |
2.75±0.09 |
0.35±0.01 |
3.03±0.32 |
|
|
AsA |
22.20±2.04f |
5.45±0.49 |
0.239±0.010d |
2.81±0.16 |
0.38±0.02 |
3.39±0.33 |
|
|
TU |
21.87±2.05f |
5.35±0.29 |
0.224±0.011de |
2.71±0.17 |
0.36±0.02 |
3.39±0.33 |
|
|
CCC |
19.37±2.02g |
5.67±0.40 |
0.206±0.015ef |
2.64±0.16 |
0.36±0.02 |
3.46±0.27 |
|
|
Kin |
21.23±2.65f |
5.75±0.33 |
0.231±0.009de |
2.75±0.15 |
0.39±0.02 |
3.77±0.28 |
|
|
Et→Det |
No spray |
27.17±1.54e |
6.83±0.38 |
0.268±0.012c |
4.50±0.30 |
0.44±0.03 |
4.65±0.25 |
|
Water |
27.37±1.26e |
7.29±0.62 |
0.278±0.010c |
4.81±0.31 |
0.55±0.03 |
4.86±0.41 |
|
|
AsA |
35.62±2.19ab |
8.17±0.40 |
0.360±0.015b |
5.42±0.33 |
0.57±0.03 |
5.26±0.26 |
|
|
TU |
35.39±1.21ab |
8.18±0.40 |
0.356±0.017b |
5.38±0.29 |
0.59±0.03 |
5.17±0.26 |
|
|
CCC |
35.97±1.72ab |
8.23±0.37 |
0.366±0.016b |
4.82±0.29 |
0.58±0.03 |
5.49±0.25 |
|
|
Kin |
34.80±2.59b |
8.35±0.62 |
0.351±0.019b |
5.56±0.32 |
0.59±0.03 |
5.56±0.41 |
|
|
|
SE (Et × FS) |
1.545* |
ns |
0.013** |
ns |
ns |
ns |
*, P < 0.05; **, P < 0.01; ns, P >
0.05. Means sharing same alphabet differ non-significantly (P > 0.05)
In non-etiolated seedlings, all the foliar spray of PGRs
increased AsA contents while the KIN and TU (9%
increase with each) were more effective. Etiolated seedlings indicated severely
reduced (by ~60%) AsA contents. All foliar
spray treatment on de-etiolated seedlings led to regain of the AsA contents but the KIN (24% regain) was highly promising
(Table 2). The NIA contents were also increased with foliar spray treatments
while AsA indicated the greatest increase (10%) in
non-etiolated seedlings. Etiolated seedlings exhibited nearly 50% decline in
the NIA contents despite foliar spray of PGRs. However, in de-etiolated
seedlings, NIA contents improved with all PGRs and the greatest increase (~34%)
was noted with TU spray (Table 2). In non-etiolated seedlings RIB contents
increased with PGRs spray but the highest increase (15%) was noticed with
foliar spray of KIN. In etiolated seedlings reduced RIB decreased despite
foliar spray of PGRs, but KIN spray led to a lowest decline (33%). De-etiolated
seedlings exhibited a regain in NIA content with PGRs spray while KIN (with 20%
regain) was highly effective (Table 2).
Trendlines
and correlations
Trend-lines were set and correlations were drawn of the
seedling dry weight with the physiological attributes under all three
condition based on the PGRs foliar spray (Fig. 4). In non-etiolated seedlings, Pn, PHE, FLA,
ANT, NIA and RIB were positively correlated while H2O2
and MDA were negatively correlated. In etiolated seedlings, none of the
physiological attributes was significantly correlated with seedling dry weight.
For de-etiolation seedlings, all the physiological characters were positively
correlated except Ci, H2O2
and MDA which were negatively correlated. However, AsA
was not correlated with shoot dry weight (Fig. 4).
Discussion
It is well established that light triggers almost all
the plant phenomena after either its absorption or by acting as a signal (Lau
and Deng, 2010). However, the clear evidence on the role of light in modulating
the PGRs action is not yet available. In this study etiolation for six days
resulted not only in the substantial elongation of seedlings but also led to
loss of seedling dry mass, eventually giving a high shoot elongation/root dry
weight ratio. Compared to unsprayed or water-sprayed seedlings the foliar spray
of PGRs partially nullified the influence of etiolation, which indicated the
effectiveness of PGRs in a regain of dry weight and a decline in the elongation
of seedlings. Although all the PGRs were effective in producing these changes,
KIN and CCC were quite more effective than the others (Fig. 1). It is reported
that light integrates the phytohormonal signaling by phytochrome interacting
factors (PIFs) in incorporating the morphogenetic changes (Lau and Deng 2010).
These data suggested that although light has its own basic role in modulating
the plant growth (Hossain and Kamaluddin
2011; Liu et al. 2017), it appeared
to synergistically improve the plant efficiency with the foliar spray of PGRs
in non-etiolated and de-etiolated seedlings.
Photosynthesis comprises two main of reactions; light
reactions (to harness solar energy with photosynthetic pigments and generating
reducing powers) and dark reactions (for the production of assimilates using CO2,
water and reducing powers). In this study we found that all the PGRs enhanced
the biosynthesis of Chl
and Car but the effects of PGRs were
well pronounced on de-etiolated seedlings. Among the PGRs foliar applied in
this study, KIN and CCC were more effective than TU and AsA
in improving pigment contents (Fig. 2) and leaf gas exchange (Table 1). For
plants KIN is a natural growth promoter (Klíčová
et al. 2004; Petrasek
et al. 2019) while CCC is a synthetic
growth retardant (Kumar and Sharma 2019; North et al. 2010). During the biosynthesis of Chl, the synthesis of
5-aminoleuvolinic acid (5-ALA), a committed step in the Chl biosynthesis (Eckhardt et al.
2004;
%20801-810_files/image008.png)
Fig. 4: Trendlines and Pearson’s correlation coefficients (r) of shoot dry weight with physiological variables of control
(non-etiolated; ●), etiolated (%20801-810_files/image010.png){kind=small}
) and de-etiolated (∆) seedlings of maize under foliar spray of plant growth regulators (n=6)
Steccanella et al. 2015),
is either independent of light (Meller and Grassman 1982) or its synthesis is stimulated under low
light intensity and inhibited under high light intensity (Aarti
et al. 2007). The cytokinins
(including KIN) enhance the biosynthesis of chlorophyll by promoting the
synthesis of its precursor (5-ALA) when applied exogenously (Jamei et al.
2008). Likewise, PGRs including CCC, TU and AsA are
also reported to improve the leaf Chl and Car contents
(Borraccino et
al. 1994; Wahid et al. 2017; Arshad et al.
2019), but likely mechanisms of action of these PGRs in either improving the
synthesis or curtailing the loss of chlorophylls in etiolated and de-etiolated
plants are obscure yet. In this study, the etiolated seedlings measured for the
photosynthetic pigment contents revealed that less of Chl a and more of Chl b contents
were reduced resulting in an enhanced Chl a/b ratio, while Car was severely declined in unsprayed etiolated maize seedlings.
The foliar spray of PGRs was poorly effective in improving photosynthetic
pigment decline during etiolation. However, during de-etiolation there was a
swift de novo synthesis of these
pigments, reaching about the level of non-etiolated plants in six days
especially with the foliar of KIN and CCC (Fig. 2). The presence of strong
positive correlations in the photosynthetic pigment contents and seedling dry
weight of de-etiolated seedlings validated our standpoint (Fig. 4). Although
this study has clearly revealed the positive effects of foliar spray of the
PGRs, further studies are mandatory to elucidate the mechanisms of these PGRs
in improving the photosynthetic pigment contents during
etiolation/de-etiolation swapping.
In this
research, gas exchange properties of leaves indicated that Pn, E, and gs
were severely reduced due to etiolation, while Ci was increased (Table 1). Shifting of the plants to light chamber
for de-etiolation quickly improvised these attributes, while Ci was reduced (Fig. 2). In etiolated seedlings, hampered leaf gas
exchange is likely due to depletion of the levels of reducing powers with loss
of photosystems (PSI and PSII) in etiolated seedlings, which
showed a quick regain (especially of PSII)
upon greening (Baker and Butler 1976). This revealed that during etiolation the
plants suffered great limitation in the availability and utilization of
resources to carry out leaf gas exchange in the production of photoassimilates, while the foliar spray of different PGRs
was partially helpful in the sustained photosynthetic pigment content and leaf gas
exchange. These changes were similar to those observed in the abiotically
stressed plants (Cassola et
al. 2019), hence supporting the view that
etiolation is also a stress factor. The positive correlations of seedling dry
weight with the Pn,
E and gs while negative with Ci in de-etiolated seedlings but not in
non-etiolated and etiolated seedlings (Fig. 4) witnessed that upon
exposure to light the effectiveness of the PGRs was evident from a gain in dry
mass (Fig. 1) upon being relieved from light limited condition.
To
substantiate further that whether or not absence or low-availability of light
produces the effects like other abiotic stresses in the etiolated seedlings,
the measurements were made for changes in the level of H2O2
(a representative ROS) and MDA (a membrane lipid peroxidation product) to
determine the extent of oxidative damage in non-etiolatied,
etiolated and de-etiolated seedlings. The results revealed no great changes in
both H2O2 and MDA content of non-etiolated plants with or
without foliar spray treatments. However, when the seedlings were shifted to
darkness for etiolation, non-sprayed or water sprayed seedlings exhibited
highest accumulation of both H2O2 and MDA, while PGR’s sprayed
seedlings manifested quite lesser (10 to 15%) decline in the amounts of both
these metabolites. However, de-etiolated plants revealed a substantial
reduction (more or less 100%) in the level of both H2O2
and MDA, which was similar to or lesser than the level measured in
non-etiolated plants (Fig. 3). The accumulation of both these metabolites in
etiolated maize seedlings was similar to that accumulated in the abiotically
stressed plants (Turan and Tripathy
2013; Hameed et
al. 2014). Although foliar spray of PGRs was lowly effective in reducing
the concentration of H2O2 and MDA, we believe that
etiolation causes oxidative damage and weak cell walls and elongated internodes
phenotypes may be the negative consequence of this phenomenon (Sinclair et al. 2017). Although no reports are
available on the effects of ROS on the cell wall properties in plants, it is
reported that high ROS levels were responsible for damage to the cell wall of Candida albicus
and Aspergillus niger
(Athie-García et
al. 2018). The diminution of both these metabolites in de-etiolated
seedlings (exhibiting reversal to normal growth) and their tight inverse
correlations with dry weight of de-etiolated seedlings (Fig. 4) further
supported our notion. Nevertheless, this aspect needs to be investigated
comprehensively in plants.
To
substantiate that the antioxidative defense is modulated in maize seedlings
during etiolation/de-etiolation switch over in this study, the levels of
phenolics (PHE, FLA, ANT) and vitamins (AsA, NIA,
RIB) were measured (Table 2). It is established that antioxidants are
synthesized in abiotically stressed plants to scavenge the ROS and improving
the functional properties of cellular membranes (Zhishen et al.
1999; Jaleel et al. 2009; Derakhshani et al. 2017; Derakhshani
et al. 2017). Our data showed that
levels of the measured phenolics and vitamins remained fairly constant in
non-etiolated seedlings; declined markedly in etiolated seedlings, but showed a
regain in de-etiolated seedlings (Table 2). Taken together, changes in the
oxidative stress and antioxidants accumulation, it was found that all these
changes were consistent but contrary to each other (Fig. 3). This indicated
that etiolation induced the oxidative damage, and reduced the ability of maize
to synthesize antioxidants (phenolics and vitamins). However, lack of
association of seedling dry weight with phenolics and vitamins in etiolated
seedlings and strong positive association of these attributes in de-etiolated
seedlings (Fig. 4), revealed the interactive role of light and the PGRs in the
synthesis of antioxidants (phenolics and vitamins) and seedling development
during de-etiolation. This clearly supports our view that light is pivotal in
regulating the plant phenomena with the foliar spray of the PGRs.
Conclusion
The hypothesis that light clearly augments the PGRs
action in maize seedlings during de-etiolation was accepted based on the
improvements occurring in the leaf pigment contents and gas exchange
properties, reduced oxidative damage and improved phenolics and vitamin
contents. These findings were further strengthened with the establishment of
significant correlation between shoot dry weight and metabolic attributes of
maize seedlings, which were significant in the de-etiolated seedlings due to the
foliar spray of PGRs. Among the PGRs, the KIN was the most effective followed
by CCC, TU and AsA. The molecular mechanism of action
of these PGRs during etiolation/de-etiolation transitions warrants further
studied.
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