the brought to the laboratory and washed thoroughly

the
ATP-dependent removal of inhibitory sugar phosphates (Portis et al.  2008). A significant
reduction (Mate et al. 1993; Hammond et al. 1998) or absence of RCA (Portis 1992) considerably impairs the photosynthetic
process in higher plants. The presence of RCA in various plants provides
evidence that activation of Rubisco in
vivo requires a specific chloroplast protein in order to occur at
physiological levels of CO2 and RuBP (Salvucci et al. 1985; Abouseadaa
et al. 2015). By modulating the activation
level of Rubisco in response to light
intensity, RCA exerts an important regulatory control on photosynthesis. Furthermore,
various studies have reported different numbers of RCA polypeptides in maize,
from one to three; the molecular masses of these polypeptides are approximately
41, 43, and/or 45 to 46 kD, respectively (Crafts-Brandner and Salvucci, 2002; Vargas-Suárez et al. 2004; Ristic et al. 2009). The
present study aimed to evaluate the biological effects of ULAE on growth
parameters (shoot length, root length, fresh weight, dry weight, and seedling
length), minerals, biochemical constituents (chlorophyll content, the activity
of ribulose bisphosphate carboxylase (Rubisco)), and Rubisco activase (rca1) gene in seedlings of Zea mays. The SWE obtained from Ulva
lactuca was applied as a foliar
spray or directly added to the growth medium of maize plant cultivated in a hydroponic
system.

Materials
and Methods

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Collection of Seaweed Materials

Marine algae Ulva lactuca was collected
manually from Abu-Qir coast, Alexandria, Egypt during October 2015. The algae was then washed thoroughly with
filtered seawater many times to remove epiphytes and sands, then was brought to
the laboratory and washed thoroughly in tap water 3 to 4 times to remove excess
salt.

Preparation of Seaweed extract

The
washed and cleaned seaweed was shade-air dried for 2–4 days followed by oven drying for 12 h at 60 °C. The oven dried seaweed was hand
crushed and finely powdered with mixer-grinder. Of the dried material, 100 g
was extracted with 1000 mL of distilled water for 24 h. The contents were then
filtered through a double-layered muslin
cloth. The filtrate thus obtained was considered as 100% seaweed
aqueous extract. A 10%
extract concentration was prepared using double distilled
water and stored at 4 °C.

 

Plant material and experimental design

Zea
mays
grains (M10) were procured from the Crop Institute, Agricultural Research
Center, Giza, Egypt. The grains having the uniform size, shape, color and weight were selected for the study. Ten grains were arranged in 18-cm diameter Petri dishes
on two layers of filter paper (Whatman number 1) under normal laboratory
conditions at 20–23 °C day/14–16 °C night. Afterward, 15 mL
of distilled water was then added. Before sowing, the grains were surface
sterilized by soaking for 2 min in 4% sodium hypochlorite, then, rinsed four
times with double distilled water. Seven–day old seedlings were transferred to the
growth
units which consisted of 4 polyethylene tubes (5 cm diameter, 51 cm length).
Each tube had an out and inlet in order to circulate
the nutrient solution (Fig 1). The capacity
of each tube was 1500 mL with 31 pores (8 mm) distributed in two alternation
lines, where the seedlings should be settled. The micropipettes plastic tips
were used to support the seedlings during growth and when they were harvested.
An air pump terminal with a flow rate of 200
mL/min was used to aerate and circulate the
solution. The nutrient solution was completely renewed every three days.
The experiment was performed under normal laboratory conditions (20  ±2°C temperature, 75  ±2% relative humidity, and 14/10 h light/dark
photoperiod).

The
experimental design

Four
treatment groups were designed for the application of ULAE in the growth medium
of maize plant cultivated in the hydroponic system. The first group of
seedlings was grown in medium containing
50% of half-strength Hoagland’s solution
and 50% of ULAE (50% U + 50% H). The second group of seedlings was grown in a medium containing 25% of half-strength Hoagland’s solution and 75% of ULAE
(75% U + 25% H). Whereas, the third group of seedlings was grown in 100% half-strength Hoagland’s solution and were not treated with ULAE (control)
and seedlings of the fourth group were grown in ULAE alone (10%) served
as a positive control. In a parallel experiment,
four foliar applications consisting of
three different ULAE concentrations (0.5%, 1%, and 5%) and one control treatment (no spray) were given
to seedlings grown in the hydroponic system with half-strength Hoagland’s solution. About 50 mL of different
concentrations of the extract or water (for the control treatment) was given at
3 days intervals up to 14 days. After 14 days, the homogenous seedlings were
carefully collected from each treatment, and then gently blotted with filter
paper. The growth parameters such as root length, shoot length, fresh and dry
weight, and seedling length were recorded. The biochemical parameters including
chlorophyll a, b, and total chlorophyll content, carotenoid and Rubisco were
also analyzed. For the determination of seedlings, the dry weights samples were
dried at 65oC till constant weight. The experiment was carried out
in triplicate.

Determination of total
protein

Protein content was
estimated according to the method
described by Hartree (1972).

Measurement of
Photosynthetic Pigments. Photosynthetic pigments, chlorophyll a, chlorophyll b
and carotenoids were extracted and determined from new and fully expanded young
leaves according to the method of William and Bloom (1985).

Analyses
of photosynthetic pigments

Leaf
tissue was added to a pre-chilled mortar in an ice bath and were ground with
pestle in Chlorophyll extraction from harvested leaves was done using 4ml of
N,N-dimethyl formamide (DMF) for 24hs at 4°C. The samples were subsequently
kept in a deep freezer for further spectrophotometric analysis. Chlorophyll was
quantitatively determined using spectrophotometry at wave lengths of 646.8 and
663.8nm (Porra, 1991).
Total caroteneoids content was calculated according to Moran (1982) and related to leaf
weight.

 Chl. a = 12.00 ·Abs.663.8-3.11·Abs.646.8

 Chl. b = 20.78 ·Abs.646.8-4.88·Abs.663.8

 Chl. a+b = 17.67 ·Abs.646.8+7.12·Abs.663.8

Total
caroteneoids = 1000 Abs.470 –0.89 (Chl. a)-52.02(Chl. b)/ 245

 The contents were expressed as mg Chl or
carotenoids g?1 fresh weight (FW).

 

Protein Extraction and Relative Amount of Rubisco
Enzyme Estimation

The 2nd
leaf of ten plants was used for the extraction
of protein and subsequent enzyme activity and protein amount measurements. The
leaves were coarsely ground in liquid N2 with a mortar and pestle. Then, 600 mg of this coarsely ground leaf
tissue was homogenized in 6 mL of buffer containing of 0.1 M (Tris hydroxymethyl
aminomethane)-HCl (Tris-HCl, pH 7.5), 5 mM ethylene glycol-bis (?-aminoethyl ether)-tetraacetic acid (EGTA), 1 mM
phenylmethylsulfonyl fluoride (PMSF), 1 ?M pepstatin, 2 mM dithiothreitol
(DTT), 5 mM MgCl2, 10 mM NaHCO3, 1.5% (w/v)
polyvinylpolypyrrolidone, and 5 ?g/mL leupeptin. Soluble and insoluble
fractions were separated using centrifugation at 17,608 g at 48 °C for 15 min.
Then, 2.5 mL of protein-containing supernatant was subsequently desalted on a
Sephadex G25 column (Pharmacia PD–10, Orsay, France) in darkness at 48°C with
elution buffer composed of 0.1 M Tris-HCl (pH 7.5) and 2 mM DTT. The desalted
protein extract was immediately used for the determination of enzyme activities. Proteins present in the
remaining supernatant (about 1 mL) were then precipitated according to Leitao et al. (2007). After
centrifugation (17608 g, 48 °C, 10 min), the pellets were resuspended in 200 ?L
of extraction buffer. Relative amounts (%) of Rubisco (SSU and LSU) were
measured using HPLC analysis (C18 column)
according to Leitao et al. (2007).

Protein concentrations in both desalted and
precipitated extracts were determined using the method described by Hartree (1972).

Rubisco activase 1 gene (rca1) detection technique

Total DNA was extracted from
new fully expanded young leaves using GeneJET Genomic DNA purification kit following
the manufacturer’s protocol (Thermo- Scientific)

PCR
Amplification of rca1

 The PCR was
carried out in 25 µL reaction tubes containing 50 ng of template DNA, 1 pmol of
each primer, and 1X PCR Master mix.
The reaction mixtures were subjected to the following cycling steps: 94 °C for
5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 52
°C   for 30 s and extension at 72 °C for
30 s. The thermal profile ended with a final extension at 72 °C for 10 min (Abdel Khalik et al., 2013;  Munshi and Osman 2010; Abdel Khalik and
Osman 2017). The
PCR product was stored at 4°C. The PCR products were analyzed using 0.45, 0.6
and 0.75% agarose gel electrophoresis
(SeaKem LE agarose, Cambrex, gels for genomic and amplified DNA) and visualized under UV fluorescence after staining
with ethidium bromide and the images were acquired using Gel Doc XR+
Imaging system (Bio-Rad Laboratories Inc., Germany). The following primer set was
used for the PCR amplification of rca1,
forward primer, 5′-CGAATGGCTCATTAAAACAG-3′ and reverse primer 5′-CCAACTACGAGCTTTTTAAC
-3′. Gene
specificity of primers was confirmed throughout BLAST searches in public
databases

Statistical
Analysis

The
quantitative data were analyzed by one-way analysis of
variance (ANOVA) and Student’s t-test
using COSTAT 2.00 statistical analysis
software manufactured by CoHort Software Company (Zar, 1984).
The differences in the treatment means were compared using the Least
Significant Differences (LSD) test to evaluate the significant differences of
the data at 0.05 probability level.

Results and Discussion

Determination of mineral composition of Ulva lactuca

The essential minerals such Calcium (Ca), Potassium (K), Zinc
(Zn), Magnesium (Mg), and Iron (Fe) are present in significant amounts in
U. lactuca (Table
1). Among these elements, Ca (3255.86
ppm) is the most abundant element in the U. lactuca
seaweed, followed by Potassium (287.9
ppm). The other elements are Mg (90.89 ppm), Zn (79.79 ppm) and Fe (71.17 ppm).
The high mineral content of U. lactuca
seaweed is due to the consumption of the nutritive elements in the medium,
where the algae grows.

Growth
parameters

Shoot
and root length

The shoot length of seedlings grown in Hoagland
solution (control treatment) was greater (21.9 cm) as compared to seedlings
grown in 100% ULAE (17.7 cm). A mixture of 50% H + 50% U did not affect the shoot
length; however, the maximum shoot length (24.8 cm) was found in seedlings
treated with 25% H + 75% U. Similarly ,the  root length was also affected by different
treatments,  an increased root length of
27.4 cm and 22.8 cm was observed in seedlings treated with a mixture of 25% H +
75% U  and seedlings grown only in 100%
ULAE, respectively, when compared to 22.8 cm root length of the control (Fig. 2).
In foliar application experiment, the use of ULAE significantly promoted the growth
and physiology of Zea mays. The treatments applied as
a foliar spray of ULAE at 0.5% or 1% displayed an increase in the growth and biochemical parameters. However,
the inhibitory effect was also observed when seedlings were sprayed with the
higher concentration of ULAE (5%). A significant
increase in the seedling length was observed when ULAE applied
as a foliar spray at 0.5%
and 1% treatment concentration, which may be attributed to the increase in root
length rather than shoot length. The maximum effect was found with 0.5%
treatment where the seedling length reached 51.4 cm compared to the control (44.7
cm). However, there was a reduction in the seedling length when the seedlings were
sprayed with 5% ULAE, the
reduction was mainly due to the reduction of root rather than shoot length.

Fresh and dry weight

The fresh weight of the control seedlings was 3.45 g. The
seedlings grown in 100% ULAE and 50% H+50% U had relatively lower fresh weights
than the control. The seedlings grown in 25% H + 75% U had a significantly higher
fresh weight (4.15 g) compared to the control and the percent increase was
16.8%.

The different
applications of ULAE treatments had no effect on the dry weights of seedlings (Fig.3). The differences in fresh and dry weights showed similar
responses to foliar applications of ULAE as of seedlings length. A
decrease in the fresh weight (2.84 g) of seedlings was observed with the higher
concentration of ULAE (5%) compared to the corresponding control (3.45 g); however,
an increase in fresh weight was found in other
two treatments. The maximum increase was obtained at a concentration of 0.5% of
ULAE where fresh weight reached 4.25 g with a percentage increase of 18.8%
compared to the control. The dry weight of seedlings was significantly enhanced
by all treatments of ULAE
applied by foliar spray when compared to the control plants.

Leaf area

The highest leaf area of 16.4 cm2 of Zea
mays was recorded for the seedlings receiving H25%+U75% treatment, whereas,
seedlings sprayed with 5% extract had the lowest leaf area (10.33 cm2).

Photosynthetic
pigments

Total photosynthetic pigment content in the control
seedlings was 27.88 mg/g. However, a significant decrease in total
photosynthetic pigment content of Zea mays seedlings was recorded for those
received different treatments (Table 2). Furthermore,
the total
photosynthetic pigment content of seedlings sprayed with 5% ULAE was significantly
reduced, and the percent reduction observed was 36% as compared to the control.
This
reduction in total photosynthetic pigment content of the seedlings sprayed with
different concentrations of ULAE could be attributed to the decrease in both Chlorophyll
a and Chlorophyll b. The percentage of reduction in Chlorophyll a was higher than that of Chlorophyll b, it was
42% and 34% in seedlings sprayed with 1% and 5% of ULAE, respectively when compared to the corresponding
control value. The percentage of reduction in Chlorophyll b reached 11% in 1% ULAE and 27% in 5% ULAE
treatments, respectively. Similarly, carotenoid contents decreased
significantly in response to different treatments.

Elemental content

The effects of ULAE on the mineral composition of Zea
mays are presented in Table 3. The maximum concentration of Ca (2212.18
mg/g) and Fe (219.3 mg/g) was recorded in seedlings that were grown in 100% U
with a percentage increase of 74% and 66%, respectively, as compared to the
control. However, the concentration of copper (Cu) was significantly reduced in
all treatments tested. The seedlings that received foliar spray with 1% ULAE exhibited
the highest content of K, Mg, Na, and Cd as
compared to the corresponding control. A significantly increased concentration of
Ca was recorded on the application of
ULAE compared to the control, where the percent increase was 60%, 59%, and 55% in seedlings sprayed with 0.5, 1, and 5% ULAE. respectively.

Protein Profile

The
protein profile of 14-day old Zea mays
seedlings treated with ULAE was analyzed and the results are shown in Figure 5. The soluble protein fraction was relatively
higher than the insoluble fraction particularly in seedlings sprayed with different
concentrations of ULAE. A decrease in the insoluble protein fraction was
recorded in the seedlings treated with 0.5% ULAE, the decrease recorded was
56.2%, when compared with the corresponding control. On the other hand, the seedlings
grown in 50%U+50%H showed the highest total protein value among all the
treatments, which was due to an increase in the insoluble fraction compared to
the soluble one. The percentage increase in the insoluble fraction of those
seedlings was 36% compared to the corresponding control.

Rubisco and Rubisco activase  

Among all treatments tested, a higher concentration of
Rubisco enzyme was recorded in the seedlings treated with 100% U and those
sprayed with 5% ULAE, as compared to the control. The highest Rubisco activity was
observed in seedlings grown in U 100% (Fig. 6). 

Rubisco
activase transcripts from Zea mays leaves tended to be less abundant in
all treatments compared to the control, suggesting that Rubisco from Zea
mays leaves might be less activated. The analysis of the final preparation
by agarose gel identified two bands with approx. 372 and 274 bp (Fig 7). 
Conversely, the supplementation of 100% U tended to promote the
accumulation of Rubisco activase transcripts. Even if measurements of
steady-state mRNA content do not necessarily reflect the rate of translation or
protein synthesis (Boschetti et al., 1990), our results suggest that
application of ULAE to the growth medium may either up- or down-regulate the
carboxylation activity catalysed by Rubisco by modifying the quantity of the
enzyme and/or by changing regulation of its activity. The presence of Rubisco activase explains how Rubisco can achieve and maintain a high
activation state in vivo at normal levels of CO2 in the presence
of millimolar concentrations of RuBP (Salvucci et al. 1985). The occurrence of activase in
several higher plant species provides evidence to a fundamental role of
activase in the control of Rubisco enzyme
in higher plants. It was particularly important that activase was detected in
maize, a C4 plant, since the CO2 concentration is higher at the site
of carboxylation (Hatch, 1971),
while RuBP is present in millimolar concentrations. Thus, the occurrence of
activase would be anticipated if the major function of activase is to maintain Rubisco in the activated state and prevent RuBP
deactivation by catalyzing the activation of the tight binding enzyme-RuBP
complex.

 The present study investigated the effect of different concentrations of ULAE
added to the growth medium or applied as a foliar spray on the seedling of Zea mays. The study showed an increase
in the vegetative growth parameters by the application of seaweed extract of Ulva lactuca. The results of the study coincided with those of the earlier studies on
Phaseolus vulgaris L. (Kocira et al. 2013).
The use of ULAE as
a foliar spray significantly promoted growth and physiology of Zea mays.
There was a significant increase in the growth and biochemical constituents of Zea mays when the seedlings were sprayed
with 0.5% or 1% of ULAE, while at the
higher concentration seedlings growth was inhibited. Similar
observations were also reported in earlier studies where seaweed extracts were applied as sprays under
controlled experiments resulting in the increased height and improved root
growth in tomatoes (Zodape et al 2011). Similar results were also reported in spinach, Vigna mungo, and tomatoes (Featon
by-Smith and van Staden 1983; Ganapathy and Sivakumar 2013, Hernández-Herrera et al. 2013; Alothyqic et al. 2016). The biochemical constituents including total
photosynthetic pigment content of seedlings sprayed with 5% ULAE were significantly reduced, and the percent
reduction recorded was 36%. This
reduction in total photosynthetic pigment content of the seedlings sprayed with
different concentrations of ULAE could be attributed to the decrease in both chlorophyll a and chlorophyll b. An increase in
the chlorophyll content in the leaves after the application of extracts from
seaweeds was reported by Blunden et al., (1996);
however, a negative effect on this trait was recorded by Venkataraman Kumar and
Mohan (1997). Further studies
on the field applications are needed to provide practical recommendations on
the positive effects of seaweed extracts on the growth and development of
maize. 

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