Introduction
Apple (Malus ×
domestica Borkh) is one of the most extensively cultivated fruit trees
worldwide (Reig et al. 2018). China has taken a prominent position in the
apple industry of the world with an average production of 39.70 million tons,
and the cultivated area is 2.41 million ha that represents 49% and 46% of the world’s
share, respectively (FAO 2016). Commercial fruit plants typically consist of
two separate genotypes: aerial part (scion) and the underground portion
(rootstock) make them complex objects for studying shoot-root communications (Forcada et al.
2014; Nawaz et al. 2016; Hayat et al. 2019). Dwarfing rootstocks
are necessary for early and high-density apple plantations (Tworkoski and Fazio 2015). These rootstocks
influence many biochemical and physiological parameters of grafted scions (An et al.
2017; Adams et al. 2018). The
physiological mechanisms of how rootstocks influence scion vigour are complex
and are not fully understood.
Physiological
and biochemical attributes that help the rootstocks to control plant vigour
have generally focused on hormone biosynthesis (Hooijdonk
et al. 2011; Tworkoski and Fazio
2015), nutrient uptake (Amiri et al. 2014; Kviklys et al. 2017; Khan et al. 2020), carbohydrates (Gemma and Iwahori
1998; Foster et al. 2017),
hydraulic conductance (Basile et al. 2003b; Solari and DeJong 2006), phenolic contents (Yıldırım et al. 2016) and alteration in anatomical structures (Saeed et al.
2010; Tombesi et al. 2011); and even within a single species (Malus
pumila), evidence exists supporting different mechanisms of scion control (Gregory et al.
2013). Size-controlling
rootstocks could affect the morphological parameters of grafted plants
differentially, which includes tree volume, intermodal length, branch
composition, trunk cross-sectional area (TCSA) and fruit yield (Gjamovski and Kiprijanovski
2011; Hooijdonk et al. 2011; Karlidag
et al. 2014).
Hormonal
regulations have been suggested as a mechanism by which rootstocks modify scion
vigour by altered shoot–root–shoot chemical signaling (Pérez-Alfocea et al. 2010; Ghanem et al.
2011; Song et al. 2016; Nawaz et al. 2017). Lower concentrations of growth-promoting hormones
such as auxin, cytokinin and gibberellin and the higher concentration of
growth-inhibiting hormones such as abscisic acid in dwarfing rootstocks have
been studied in fruit plants. Hormones are well known for controlling tree size
(Gregory et
al. 2013). The rootstock or interstock induced dwarfing effect is
due to the alterations in gene expressions linked with hormonal metabolism and
transduction, which in turn may regulate the balance of endogenous hormones in
scion (Aloni et al. 2010; Tworkoski
and Fazio 2016). Auxin
synthesis primarily occurs in the leaves and roots. YUCCA monooxygenases
catalyze the conversion of indole-3-pyruvate to IAA that has a crucial role in
auxin biosynthesis (Zhao et al. 2001; Won et al.
2011). Isopentenyl transferases are the essential enzymes that play a
vital role in cytokinin synthesis. Gene expressions of IPT directly regulate endogenous cytokinin concentration (Singh et al.
1992; Samuelson and Larsson 1993; Takei et
al. 2004).
Several reports
suggest that rootstocks influenced the scion nutrient accumulation in apple and
peach plants and the alterations among scion-rootstock communications were
linked with nutrient uptake capacity because of their different route structure
(Abrisqueta et
al. 2011; Kviklys et al. 2017).
Rootstock-scion interactions induce apparent changes in absorption and
transport of mineral nutrients to the scion by the alteration of the root structure.
However, rootstock performance was not consistent among sites and varied over
time (Al-Hinai and Roper 2004).
Rootstocks
that reduce scion growth are influence by root morphology, root spreading
features, and their association with scion (Ma et al. 2013). Dwarfing rootstocks
for fruit plants have been used for a long time; the underlying mechanism of
rootstock-induced dwarfing is still unknown (Foster et al. 2017). The selection of dwarfing rootstocks and screening
for superior rootstocks from apple hybrid seedlings are necessary for the
breeding of dwarfing rootstocks. This study consists of the measurement of
physiological and biochemical indicators for the different plant parts of young
apple trees. This study aimed to obtain suitable indices that could be used to
predict the degree of dwarfing at the early stage of apple plant growth.
Furthermore, we also studied the hormonal levels and the relative expression of
hormone-related genes controlling rootstock induced dwarfism in apple plant.
Materials and Methods
Plant
materials and culture conditions
The experiment was performed
at the Horticultural Laboratory of China Agricultural University, Beijing,
China (Longitude 116°24' E, latitude 39°54' N) during March-September, 2018. In
March 2018, 1-year old plants of “Red Fuji” grafted onto four rootstocks viz.,
Malling 9 (‘M.9’, dwarfing), Malling 26 (‘M.26’, semi-dwarfing), ‘Chistock-1’ (Malus xiaojinensis, semi-dwarfing) and
‘Baleng’ (Malus micromalus,
vigorous) were grown in a 30 cm diameter pots containing a blend of
garden soil, nursery substrate and sand (3:2:1 v/v) and grown under greenhouse
conditions. The plants were irrigated
twice on each day, fertilized (1.75 g of 20 N-8.8P-16.6 K w/w/w per tree) on a
monthly base. The experiment was carried out in Randomized Complete Block
Design (RCBD) and three biological replicates were made (every replicate
containing two plants). A total of 120 plants were used in this study.
Determination
of morphological characteristics of grafted plants
From May 2018, observations
were taken regarding vegetative growth such as plant height (cm), shoot length
(cm), leaf number, node number, and intermodal length at 20 days’ interval for
each scion-rootstock combination. A digital caliper was used to measure scion
diameter (mm) and converted into TCSA using the given formula: TCSA = (d/2)2
(Rahmati et
al. 2015). The leaf area (cm2) was measured by using the
LI-3100C Area Meter (Li-Cor, Inc., Lincoln, Nebraska, U.S.A.). The measurement
of root morphology traits, healthy plants were carefully uprooted and gently
washed with water before the leaves dropped in the autumn of 2018. The data for
root morphology parameters was assessed by scanning roots using root scanner,
and analysis was done by WinRHIZO software (Regent Instruments Inc., Quebec,
Canada). The shoot to root ratio was determined by using the formula root fresh
weight/shoot fresh weight.
Photosynthetic measurements and hydraulic conductance
A portable photosynthesis system (LI-6400XT, Licor, Inc.,
Lincoln, NE, USA) was used for measuring net photosynthesis rate (Pn), intercellular CO2 concentration
(Ci), stomatal conductance (gs) and transpiration (E), of the fully expanded leaves from
9:00 to 11:30 a.m. after 45, 90 and 180 days after grafting (DAG). All
measurements were performed under the given environmental factors: leaf
temperature 25 ± 2°C; relative humidity (RH) 65 ± 5%; photosynthetic photon
flux 1200 μmol m-2 s-1
and external CO2 concentration 400 μmol mol-1. The leaf water potential (ψleaf)
was estimated on uniform fully expanded mature leaves on the same day as gas
exchange measurements by the usage of a pressure chamber (Soil-moisture
equipment Corp, Santa Barbara, C.A., U.S.A.). Tensiometers were installed at a
deepness of 15cm to measure the matric potential (ψsoil) of the
pots, as described previously (Qiu et al. 2016). The evaporative flux method was used to estimate the Soil -to-leaf
hydraulic conductance (Ksoil-leaf).
Ksoil−leaf = E/
ψsoil− ψleaf.
Phytohormone
and carbohydrates determinations
Leaf and root samples for endogenous hormones were collected from different
scion-rootstock combinations at 45, 90, and 180 DAG. The concentration
of zeatin riboside (ZR), indole-3-acetic acid (IAA), abscisic acid (ABA) and
gibberellin (GA3) were measured using Enzyme-linked immunosorbent
assay (ELISA) as mentioned by (Zhao et al.
2006). The quantification of
hormones was calculated based on a standard curves and expressed as ng g−1
fresh weight. Soluble sugars and starch were extracted according to the method
defined by (Filip et al. 2016). The amount of soluble sugars and starch were
reported as mg g−1 DW.
Measurement of mineral nutrition
Leaf and root samples were
harvested at 45, 90, and 180 DAG. Samples were carefully washed with deionized
water and then dried at 65°C for 48 h until gaining constant weight. Dried
samples were grounded so they can pass through a 40 mesh screen by using a
Cyclotec Sample Mill (Cyclotec 1093, Teactor, Hoganas, Sweden). The powder was
digested in a mixture of H2SO4–H2O2.
The nitrogen (N) concentration was estimated according to the Kjeldahl method (Nelson and Sommers 1980). The concentrations
of other elements of minerals (Mn, P, Ca, Mg, Fe, K, Cu, and Zn) were measured
using ICP-MS as per the methods termed by (Masson
et al. 2010). The results were
expressed on a dry matter basis: g kg−1 for macro elements (N,
P, K, Ca and Mg) and mg kg−1 for microelements (Mn, Cu, and
Zn).
Table 1: Primer sequences for the quantification of transcripts by RT-PCR
Primer sequences
Primer |
Primer
sequence |
IPT3A Fwd |
5-
GTGTTCAATCCTCACCGGCA-3 |
IPT3A Rev |
5-
GCCGATCACGGCCTAAAATG-3 |
GA20OX1 Fwd |
5- TCTCCGGTGACAAAGAAGCC-3 |
GA20OX1 Rev |
5-
GCTCTCCCCTGCTTTCCTTT-3 |
NCED1 Fwd |
5-
TCGGAGGACGACGGTTATATTC-3 |
NCED1 Rev |
5-
CCATGAAACCCGTAGGGCAC-3 |
YUCCA10a Fwd |
5-
CAAGTATCCGATCATTGAC-3 |
YUCCA10a Rev |
5-
CCTCTTATGCTGCCTATT-3 |
B-actin Fwd |
5-TGGTGAGGCTCTATTCCAAC
-3 |
B-actin Rev |
5-TGGCATATACTCTGGAGGCT-3 |
Gene expression of YUCCA10A,
IPT3A, GA20OX1, NCED1
The total RNA of leaf and root
samples were extracted and purified at 45, 90, and 180 DAG, using an RNAprep
Pure Plant Kit (TIANGEN, Beijing, China). Then the quantity and the purity of
the RNA were tested using a NanoDrop Spectrophotometer. The first-strand cDNA
was synthesized by using the PrimeScript RT reagent kit (Takara, Japan) as per
the manufacturer’s directions. Finally, gene-specific primers for RT-PCR were
designed by using the NCBI Primer-BLAST. The primer sequence list is given in
Table 1. Quantitative qRT-PCR was performed in a 7500 Real-time PCR system
(Applied Biosystems, C.A., U.S.A.) using the SYBR Premix Ex Taq kit (Takara,
Japan) as per the manufacturer’s directions. qRT-PCR was done using Quantstdio™
7 Flex Real-Time PCR System, Life Technologies™, Carlsbad, CA, USA. PCR
conditions consist of 95°C for 30 s,
followed by 40 cycles of denaturation at 95°C for 5 s and 60°C for 34
s. The standard comparative method was used to calculate relative gene
expression (Livak and Schmittgen 2001).
Leaf anatomical
characteristics
Six leaves from each treatment
combination were selected for the observations of anatomical traits (1 cm × 0.5 cm) and
fixed in a 4% paraformaldehyde/0.1 M phosphate buffer. Leaf anatomical traits
were measured as described by O'Brien and McCully (1981) and samples were
imaged using a BX51TRF fluorescence microscope (Olympus Optical Co., Ltd.,
Japan). Image-Pro Plus 6.0 software was used to measure the spongy thickness of
the leaf, as well as the xylem vessel density and xylem vessel diameter.
Statistical analysis
Data were subjected to the
ANOVA (Analysis of variance) in a complete factorial design with three sampling
points and four rootstocks using the statistical package SPSS 19.0 (SPSS Inc.,
Chicago, USA). Mean comparisons among the treatments were made using the least
significant difference (LSD) multiple comparison tests at P < 0.05.
Results
Phenotypic changes
Rootstocks affected the growth
vigour of grafted apple trees (Table 2 and 3). The shoot length of ‘Red Fuji’
apple trees grown onto ‘M.9’ rootstock was lower compared with ‘M.26’, ‘Chistock-1’,
and ‘Baleng’ rootstocks (Fig. 1). The most vigorous rootstocks (‘Chistock-1’
and ‘Baleng’) rootstocks resulted in the higher values (5.45 and 6.19 mm
respectively) in terms of trunk diameter of the scion, whereas lowest values
(5.22 and 5.35 mm respectively) were recorded in ‘M.9’ and ‘M.26’ rootstocks.
Leaf area of trees grown with ‘M.9’ and ‘M.26’ rootstocks was lower (38.31 and
42.31 cm2, respectively)
compared with more vigorous rootstocks. ‘Baleng’ rootstock produced longer
internodal lengths (1.94 cm), whereas the ‘M.9’ rootstock resulted in the
shortest internodal lengths (1.45 cm).
Table 2: Effects of different rootstocks on the growth of ‘Red
Fuji’ apple
Rootstock |
Plant height (cm) |
Trunk diameter of the scion (mm) |
Internodal length (cm) |
Leaf areas (cm2) |
Weight of above-ground part (g) |
Weight of root (g) |
Root-shoot ratio |
‘M.9’ |
90.25 d |
5.222 b |
1.45 b |
38.31 b |
515.68 c |
254.73 d |
0.494 c |
‘M.26; |
109.5 c |
5.352 b |
1.817 a |
42.31 b |
567.33 b |
321.86 c |
0.567 b |
‘Chistock-1’ |
122 b |
5.446 b |
1.58 b |
47.52 a |
597.52 b |
395.97 b |
0.662 a |
‘Baleng’ |
140 a |
6.194 a |
1.941 a |
47.83 a |
734.48 a |
507.51 a |
0.690 a |
The data are
means of three biological replicates. Different letters indicate significant
differences by LSD (P ≤ 0.05)
Table 3: Root morphological characteristics of 1- year old Red
Fuji scion cultivar grafted onto different rootstocks
Rootstock |
Average Diameter (mm) |
Root Length (cm) |
Projected Area (cm2) |
Surface Area (cm2) |
Root Volume (cm3) |
Number of Tips |
Number of Forks |
‘M-9’ |
1.58 b |
2625.96 c |
143.61 d |
451.17 d |
7.36 c |
23354.5 c |
17870.5 b |
‘M-26’ |
2.73 ab |
4333.28 c |
252.01 c |
791.71 c |
12.43 bc |
27185.5 c |
29423.0 b |
‘Chistock-1’ |
2.83 ab |
11110.13 b |
473.97 b |
1489.02 b |
16.97 ab |
75354.5 b |
102308.0 a |
‘Baleng’ |
3.86 a |
16968.46 a |
678.00 a |
2130.02 a |
22.04 a |
154002.5 a |
134317.5 a |
The data are
means of three biological replicates. Different letters indicate significant
differences by LSD (P ≤ 0.05)
The biomass of ‘Red Fuji’ trees grafted with ‘Baleng’ and ‘Chistock-1’
was markedly higher than vigour controlling rootstocks. Alterations in root
length, surface area, root volume, projected area, and the number of root forks
and tips among various rootstocks were also noticeable
different (Table 2). The ‘M.9’ plants had shorter and smaller roots, whereas
the most extensive root length was obtained for the ‘Baleng’ rootstock. The values of root morphological traits including root surface area
(2130.02 cm2) projected area (678 cm2) and the number of
forks (134317.5) and tips (154002.5), were also highest in ‘Baleng’ rootstock
compared with dwarfing rootstock (‘M.9’). The root-shoot ratios of ‘Red Fuji’
apple trees grown onto ‘Chistock-1’ and ‘Baleng’ (0.662 and 0.690 respectively)
rootstocks were also higher than ‘M.26’ and ‘M.9’ rootstocks (0.567 and 0.494
respectively).
Fig. 1: Phenotypic changes of ‘Red Fuji’ apple scion grafted
onto ‘M-9’, ‘M-26’, ‘Chistock-1’ and ‘Baleng’ rootstocks. (A) Trees grafted on M.9 rootstocks had weakest growth vigour, (B) root morphological system, and (C) shortest shoot length at different
stages of growth and development. Error bars show the standard error of three
biological replicates. Different letters indicate significant differences by
LSD (P ≤ 0.05)
Hormonal changes
‘Red Fuji’ apple trees grafted
with vigour controlling rootstocks varied in leaf and root IAA contents (Fig.
2). ‘Baleng’ rootstock enhanced leaf IAA content (88.72 ng g−1)
compared with ‘M.9’ (73.72 ng g−1) and ‘M.26’ (79.50 ng g−1)
rootstocks (Fig. 2A). Root IAA levels (44.21 ng g−1) were
lowest for ‘M.9’ rootstock compared with the ‘M.26’ (48.68 ng g−1),
‘Chistock-1’ (60.45 ng g−1) and ‘Baleng’ (69.65 ng g−1),
respectively (Fig. 2B). Leaf ZR levels (9.31 ng g−1) were
higher in the ‘Red Fuji’ apple grafted onto ‘Baleng’ rootstock, whereas lower
ZR levels were obtained for ‘M.26’ (8.21 ng g−1) and ‘M.9’
(6.82 ng g−1) rootstocks respectively (Fig. 2C). Root ZR
levels (8.61 ng g−1) were also found noticeably higher in
‘Baleng’ rootstocks compared with ‘M.9’, ‘M.26’, and ‘Chistock-1’ (by 5.81,
7.14 and 7.76 ng g−1, respectively)
(Fig. 2D).
Fig. 2: Endogenous
hormone levels in tissue of ‘Red Fuji’ apple grafted onto different rootstocks
at different sampling points (x-axis). Endogenous indole-3-acetic acid (IAA)
levels of leaf (A), and root (B); endogenous zeatin riboside (ZR)
levels of leaf (C), and root (D); endogenous gibberellic acid (GA3)
levels of leaf (E), and root (F); and endogenous abscisic acid (ABA)
levels of leaf (G) and root (H) of ‘Red Fuji’ apple grafted onto
different rootstocks. Error bars show the standard error of three biological replicates.
Different letters indicate significant differences by LSD (P ≤ 0.05)
Leaf GA3 levels (10.15 ng g−1) were markedly
higher in ‘Baleng’ rootstocks compared with other size controlling rootstocks
utilized in this study (Fig. 2E). In the root, GA3 levels (7.42 ng g−1)
were highest for the ‘Baleng’ rootstock and lowest for ‘M.9’, ‘M.26’, and
‘Chistock-1’ rootstocks (by 5.78, 5.93 and 6.13 ng g−1),
respectively (Fig. 2F). The leaf ABA levels (89.82 ng g−1)
were relatively high for ‘Red Fuji’ grafted onto ‘M.26’ rootstock followed by
‘M.9’ rootstock (87.31 ng g−1), whereas ‘Baleng’ and
‘Chistock-1’ rootstocks displayed lower levels (by 74.51 and 84.7 ng g−1
respectively) (Fig. 2G). The opposite trends were observed for root ABA levels;
whereas, the ‘M.9’ rootstock showed a lower level (44.21 ng g−1)
of ABA compared with the three other rootstocks (Fig. 2H).
Gene expression of YUCCA10a,
IPT3A, GA20-ox1, NCED1
The relative
expression of auxin synthesis gene MdYUCCA10a
was lower in the leaf of ‘Red Fuji’ apple trees grown onto ‘M.9’ rootstock
compared with trees grown onto ‘M.26’, ‘Red’, ‘Chistock-1’ and ‘Baleng’
rootstocks at 45, 90, and 180 DAG (Fig. 3A). Similarly, in the roots, the
relative expression of the MdYUCCA10a
gene was reduced in ‘M.9’ rootstock grafted with ‘Red Fuji’ compared with other
rootstocks (Fig. 3B). The relative expression levels of the IPT3A gene were found lower in the roots
and leaves of ‘M.9’ rootstock grafted with ‘Red Fuji’ apple scion cultivar and
higher with ‘M.26’, ‘Chistock-1’ and ‘Baleng’ rootstock (Fig. 3C and 3D). For
the GA20-ox1 gene, ‘Red Fuji’ apple
grafted with ‘M.9’ and ‘M.26’ rootstocks had lower relative expressions in
their leaves and roots and was comparatively higher with vigorous (‘Baleng’)
rootstocks (Fig. 3E and 3F). The expression pattern of NCED1gene was higher in the roots and leaves of ‘Red Fuji’/‘M.9’
and decreased with increasing plant size and vigour (Fig. 3G and H).
Fig. 3: Relative expression of hormones-related genes in the
leaf and roots of ‘Red Fuji’ apple grafted onto different rootstocks at 45, 90
and 180 days after grafting. Different sampling points (x-axis). Relative
expression of MdYUCCA10a in leaf (A), and root (B); relative expression of MdIPT3A
in leaf (C), and root (D); relative expression of MdGA20OX1 in leaf (E), and root (F); and
relative expression of MdNCED1 in
leaf (G), and root (H) of ‘Red Fuji’ apple grafted onto
different rootstocks. Error bars represent the standard deviations. Different letters indicate significant differences by LSD (P ≤ 0.05)
Mineral
nutrient concentrations
‘Red Fuji’ apple
cultivar grafted onto different rootstocks behaved differentially regarding
nutrient uptake and accumulation in the leaves and roots (Table 5 and 6). ‘Red
Fuji’ apple grafted onto ‘M.9’ had lower concentrations for most of the
minerals investigated. However, ‘Red Fuji’ grafted onto ‘M.9’ accumulated more
N (25.61 g kg−1) in their leaves compared with ‘M.26’ (24.55 g
kg−1), ‘Chistock-1’ (24.24 g kg−1) and
‘Baleng’ (25.22 g kg−1) rootstocks, respectively. Trees grafted onto ‘Chistock-1’ rootstock
accumulated more P (2.53 g kg−1 and 1.938 g kg−1)
in the leaves and roots compared with ‘M.9’, ‘M.26’, and ‘Baleng’ rootstocks.
‘Red Fuji’ apple trees grown onto ‘Baleng’ rootstock had higher K concentration
(14.95 g kg−1) in the leaves compared with ‘M.9’ (9.78 g kg−1),
‘M.26’ (11.64 g kg−1), and ‘Chistock-1’ (11.73 g kg−1)
rootstocks respectively. Additionally, ‘Red Fuji’ apple trees grafted onto
‘Chistock-1’ rootstock had higher Mg concentration (6.03 g kg−1)
in their leaves compared with ‘M.9’ (4.52 g kg−1), ‘M.26’
(5.33 g kg−1) and ‘Baleng’ (5.66 g kg−1)
rootstocks. Trees were grown onto ‘M.9’ rootstock accumulated less
concentration of Cu (5.45 and 5.18 mg kg−1) and Zn (22.39 and
17.52 mg kg−1) in leaves and roots compared with trees grown
onto ‘M.26’, ‘Chistock-1’ and ‘Baleng’ rootstocks. ‘Red Fuji’ apple trees grown
onto ‘M.9’ rootstock had lower values of leaf Ca concentrations (10.89 mg kg−1) compared with ‘M.26’
(14.25 mg kg−1), ‘Chistock-1’ (14.47 mg kg−1)
and ‘Baleng’ (13.78 mg kg−1) rootstocks.
Table 4: Changes in soluble sugars and starch content in the
leaves and roots of ‘Red Fuji’ apple grafted onto different rootstocks measured
at 45, 90 and 180 days after grafting (DAG)
Parameters |
Rootstocks |
Plant tissues |
45 DAG |
90 DAG |
180 DAG |
Mean |
Starch (mg/g FW) |
‘M-9’ |
Leaf |
6.1918 a |
5.3927 a |
8.3420 a |
6.7312 a |
Root |
5.6173 a |
16.356 a |
18.228 a |
13.401 a |
||
‘M-26’ |
Leaf |
5.2677 b |
3.4103 b |
5.3527 a |
4.6769 a |
|
Root |
3.3313 c |
6.0290 c |
12.954 a |
7.4382 b |
||
‘Chistock-1’ |
Leaf |
4.3130 c |
5.1520 a |
11.479 a |
6.9812 a |
|
Root |
7.8000 c |
1.3990 d |
6.7803 a |
5.3264 b |
||
‘Baleng’ |
Leaf |
5.1083 b |
5.2297 a |
4.0835 a |
4.8976 a |
|
Root |
8.2217 a |
11.242 b |
17.963 a |
12.476 a |
||
Total soluble sugars
(mg/g FW) |
‘M-9’ |
Leaf |
39.553 a |
32.391 a |
28.715 a |
33.553 a |
Root |
12.528 c |
25.702 a |
23.717 a |
20.649 a |
||
‘M-26’ |
Leaf |
27.776 b |
31.024 ab |
27.442 a |
28.747 a |
|
Root |
8.0463 d |
15.720 b |
19.614 a |
14.460 a |
||
‘Chistock-1’ |
Leaf |
25.740 c |
29.820 b |
28.295 a |
27.951 a |
|
Root |
14.288 b |
6.3380 c |
16.885 a |
12.504 a |
||
‘Baleng’ |
Leaf |
38.088 a |
31.755 ab |
23.749 a |
31.197 a |
|
Root |
17.218 a |
15.130 b |
21.551 a |
14.460 a |
Different
letters indicate significant differences by LSD (P ≤ 0.05)
Table 5: Changes in macronutrients content in the leaves and
roots of ‘Red Fuji’ apple grafted onto different rootstocks measured at 45, 90
and 180 days after grafting (DAG)
Trait |
Rootstock |
Plant part |
45 DAG |
90 DAG |
180 DAG |
Mean |
N (g kg−1) |
‘M-9’ |
Leaf |
30.36 ab |
25.80 a |
19.50 c |
25.61 a |
Root |
18.56 a |
10.96 a |
12.30 c |
14.63 a |
||
‘M-26’ |
Leaf |
29.30 b |
22.66 b |
20.76 b |
24.55 b |
|
Root |
8.90 c |
6.70 b |
13.233 b |
9.276 c |
||
‘Chistock-1’ |
Leaf |
31.00 a |
21.80 b |
20.86 b |
24.24 b |
|
Root |
5.233 d |
5.50 b |
10.60 d |
7.111 d |
||
‘Baleng’ |
Leaf |
31.23 a |
23.40 a |
22.20 a |
25.22 ab |
|
Root |
18.567 a |
10.36 a |
14.96 a |
11.91 b |
||
P (g kg−1) |
‘M-9’ |
Leaf |
2.715 c |
1.703 b |
1.901 b |
2.106 c |
Root |
1.150 c |
1.363 b |
1.430 d |
1.313 d |
||
‘M-26’ |
Leaf |
2.420 d |
2.303 b |
2.128 a |
2.283 bc |
|
Root |
1.765 b |
1.678 a |
1.960 b |
1.801 b |
||
‘Chistock-1’ |
Leaf |
2.960 b |
2.929 a |
1.701 d |
2.530 a |
|
Root |
2.033 a |
1.296 b |
2.486 a |
1.938 a |
||
‘Baleng’ |
Leaf |
3.655 a |
1.749 b |
1.816 c |
2.407 ab |
|
Root |
1.860 a |
1.244 b |
1.735 c |
1.613 c |
||
K (g kg−1) |
‘M-9’ |
Leaf |
10.06 b |
7.233 d |
12.33 b |
9.778 c |
Root |
3.396 b |
2.843 b |
5.175 b |
3.805 c |
||
‘M-26’ |
Leaf |
8.850 c |
12.90 b |
13.16 b |
11.63 b |
|
Root |
3.593 b |
4.570 a |
4.905 b |
4.356 b |
||
‘Chistock-1’ |
Leaf |
10.46 b |
9.700 c |
15.01 a |
11.72 b |
|
Root |
2.329 c |
3.466 b |
5.800 a |
3.865 c |
||
‘Baleng’ |
Leaf |
14.18 a |
15.817 a |
14.85 a |
14.95 a |
|
Root |
4.768 a |
4.768 a |
4.458 c |
4.555 a |
||
Ca (g kg−1) |
‘M-9’ |
Leaf |
9.050 c |
11.50 b |
11.96 c |
10.89 b |
Root |
10.00 ab |
7.016 b |
8.800 b |
8.608 c |
||
‘M-26’ |
Leaf |
10.20 b |
14.58 a |
17.98 a |
14.25 a |
|
Root |
9.216 b |
9.466 a |
11.83 a |
10.17 a |
||
‘Chistock-1’ |
Leaf |
10.82 ab |
14.76 a |
17.83 a |
14.47 a |
|
Root |
7.012 c |
9.375 a |
11.73 a |
9.374 b |
||
‘Baleng’ |
Leaf |
11.90 a |
13.23 ab |
16.23 b |
13.78 a |
|
Root |
10.70 a |
8.80 a |
8.783 b |
9.428 b |
||
Mg (g kg−1) |
‘M-9’ |
Leaf |
4.313 b |
5.216 b |
4.051 c |
4.527 c |
Root |
1.364 b |
1.835 b |
1.592 d |
1.597 c |
||
‘M-26’ |
Leaf |
2.692 c |
5.866 b |
6.433 a |
5.330 b |
|
Root |
1.465 b |
1.686 b |
2.136 b |
1.7628 b |
||
‘Chistock-1’ |
Leaf |
5.450 a |
7.066 a |
5.566 b |
6.027 a |
|
Root |
0.986 c |
1.895 b |
2.281 a |
1.721 bc |
||
‘Baleng’ |
Leaf |
5.475 a |
5.900 b |
5.616 b |
5.663 ab |
|
Root |
2.250 a |
2.768 a |
1.888 d |
2.302 a |
Different
letters indicate significant differences by LSD (P ≤ 0.05)
Table 6: Changes in micronutrients content in the leaves and roots of ‘Red
Fuji’ apple grafted onto different rootstocks measured at 45, 90 and 180 days
after grafting (DAG)
Nutrient |
Rootstock |
Plant part |
45 DAG |
90 DAG |
180 DAG |
Mean |
Cu (mg kg−1) |
‘M-9’ |
Leaf |
3.480 c |
6.166 b |
6.70 a |
5.4489 c |
Root |
3.350 d |
6.100 b |
6.083 b |
5.177 c |
||
‘M-26’ |
Leaf |
7.013 c |
7.866 a |
5.333 b |
6.737 b |
|
Root |
7.413 b |
9.383 a |
7.316 a |
8.037 a |
||
‘Chistock-1’ |
Leaf |
9.655 a |
7.716 a |
4.455 c |
7.275 a |
|
Root |
5.450 c |
11.21 a |
6.416 b |
7.699 a |
||
‘Baleng’ |
Leaf |
8.430 b |
4.297 c |
3.765 d |
5.497 c |
|
Root |
9.605 a |
6.500 b |
4.095 c |
6.733 c |
||
Mn (mg kg−1) |
‘M-9’ |
Leaf |
110.52 a |
64.33 a |
39.12 b |
71.32 a |
Root |
22.49 d |
43.30 b |
34.40 c |
33.39 a |
||
‘M-26’ |
Leaf |
46.77 c |
39.40 b |
31.66 d |
39.29 c |
|
Root |
31.44 a |
28.96 bc |
14.80 d |
25.07 b |
||
‘Chistock-1’ |
Leaf |
71.27 b |
55.83 a |
51.66 a |
59.59 b |
|
Root |
10.72 d |
33.00 ab |
38.71 a |
27.47 b |
||
‘Baleng’ |
Leaf |
22.49 d |
43.30 b |
34.40 c |
33.39 d |
|
Root |
28.72 b |
25.81 c |
16.72 c |
23.75 b |
||
Zn (mg kg−1) |
‘M-9’ |
Leaf |
17.21 d |
30.48 b |
25.71 b |
22.39 c |
Root |
12.46 c |
14.40 a |
19.50 c |
17.52 c |
||
‘M-26’ |
Leaf |
20.91 c |
71.50 a |
31.80 a |
46.03 a |
|
Root |
19.15 b |
23.36 a |
45.68 a |
24.77 a |
||
‘Chistock-1’ |
Leaf |
25.42 b |
26.15 bc |
28.58 b |
27.50 b |
|
Root |
19.07 b |
27.85 a |
30.93 b |
25.16 a |
||
‘Baleng’ |
Leaf |
27.80 a |
21.62 c |
17.06 c |
22.95 c |
|
Root |
31.80 a |
13.68 b |
19.42 c |
20.85 b |
Different
letters indicate significant differences by LSD (P ≤ 0.05).
Photosynthesis and gas exchange measurements
The data related to
gas exchange parameters and hydraulic conductance of ‘Red Fuji’ apple plants
grafted onto different rootstocks was affected by the rootstock (Fig. 4). The
lowest rate (13.72 mol m-2 s-1) of photosynthesis (Pn) was observed for trees growing on
‘M.9’ rootstocks, while the highest rate (by 15.29, 16.48 and 17.26 mol. m-2
s-1) of photosynthesis (Pn)
was observed for ‘M.26’ and ‘Chistock-1’ and ‘Baleng’ rootstocks respectively
(Fig. 4A). ‘Red Fuji’ apple scion cultivar grafted with ‘M.9’ rootstocks had
lower intercellular CO2 concentration (225.52
mol mol-1) and stomatal conductance (0.12 mol m-2 s-1) values compared with ‘M.26’ and
‘Chistock-1’ and ‘Baleng’ rootstocks (Fig. 4B, 4C, 4D). Compared with the
‘Baleng’, ‘Red Fuji’ apple leaves with ‘M.9’, ‘M.26’ and ‘Chistock-1’ showed
significant differences for leaf water potential. Trees grown onto ‘M.9’
rootstock had lower (-1.71 MPa) leaf water potential than trees on vigorous
‘Baleng’ rootstock (-1.28 MPa), whereas trees grafted on ‘Chistock-1’ and
‘M.26’ had intermediate values (by -1.489 and -1.563 MPa) respectively (Fig.
4E). Leaf hydraulic conductance (Kleaf)
showed substantially lower values (1.45 mmol m-2 s-1 MPa-1)
for ‘M.9’ rootstock compared with ‘M.26’ and ‘Chistock-1’ and ‘Baleng’
rootstocks (by 1.99, 2.5641 and 3.101 mmol m-2 s-1 MPa-1),
respectively (Fig. 4F).
Starch and soluble sugars
The starch content of
‘Red Fuji’ apple trees was affected by the use of the different rootstocks
(Table 4). In terms of the leaf, ‘Red Fuji’ apple trees grafted with ‘M.9’ rootstock
had higher starch contents but not statistically different from other
treatments, whereas trees grafted with ‘M.26’, ‘Chistock-1’ and ‘Baleng’
rootstocks were relatively low starch contents. Root starch contents were also
highest (13.4 mg kg−1 FW) for ‘M.9’, followed by ‘Baleng’
(12.48 mg kg−1 FW) rootstock compared with the ‘Chistock-1’
(5.33 mg kg−1 FW) and ‘M.26’ (7.43
mg kg−1 FW) rootstocks. Leaf and root soluble sugars were
relatively higher in the ‘M.9’ rootstock compared with other rootstocks,
whereas ‘M.26’, ‘Chistock-1’, and ‘Baleng’ rootstocks displayed lower soluble
sugar content.
Fig. 4: The Influence of rootstocks on the net photosynthetic
rate (A), intercellular CO2 concentration
(B), transpiration rate (C), stomatal conductance (D), leaf water potential (E) and hydraulic conductance (F) of ‘Red Fuji’ apple leaves at 45, 90
and 180 days after grafting. Error bars show the standard error of three
biological replicates. Different letters indicate significant differences by
LSD (P ≤ 0.05)
Fig. 5: Leaf anatomical characteristics of 1-year-old Red Fuji
scion cultivar grafted onto ‘Baleng’, ‘Chistock-1’, ‘M-26’ and ‘M-9’ rootstocks
Leaf anatomical characteristic
The leaf of ‘Red Fuji’/ ‘M.9’ had the lowest average
vessel density and average vessel diameter compared with ‘M.26’, ‘Chistock-1’,
and ‘Baleng’ rootstocks. Moreover, xylem area, phloem area, and xylem/phloem
ratio were highest for ‘Baleng’ rootstocks, while the other three rootstocks
recorded the lowest one (Fig. 5). ‘Baleng’ rootstock exhibited significantly
higher leaf area (47.83 cm2) and palisade thickness (81.35 um)
compared to ‘Chistock-1’, ‘M.26’, and ‘M.9’ rootstocks. Furthermore,
palisade/spongy thickness ratio (P/S) result relative to ‘M-9’ was relatively
high (1. 016), followed by ‘Chistock-1 (0.913) and ‘M-26’ (0.996), rootstocks,
whereas low values were obtained with ‘Baleng’ (0.89) rootstocks. Anatomical
characteristics revealed the differences in xylem vessel features in the leaf
of ‘Red Fuji’ grafted onto different rootstocks. The number of cortical
thickness was also lowest (335.93 um) with ‘M.9’ rootstock and highest with
‘M.26’ (343.13 um), ‘Chistock-1’ (408.69 um) and ‘Baleng’ (448.91 um)
rootstocks, respectively.
Table 7: Factor analysis of the
morphological and biochemical indices of ‘Red Fuji’ apple grafted onto
different rootstocks
Parameters |
Component 1 |
Component 2 |
Component 3 |
Plant height |
0.936 |
0.347 |
0.061 |
Shoot length |
0.932 |
0.363 |
0.002 |
Number of nodes |
0.932 |
0.348 |
-0.101 |
Number of leaves |
0.73 |
0.681 |
-0.058 |
Internodal length |
0.887 |
0.274 |
0.372 |
Trunk diameter of scion |
0.751 |
0.642 |
0.155 |
Number of root tips |
0.934 |
0.315 |
0.168 |
Number of root forks |
0.901 |
0.342 |
-0.268 |
Total root length |
0.909 |
0.415 |
-0.048 |
Root surface area |
0.925 |
0.373 |
-0.064 |
Root volume |
0.956 |
0.291 |
-0.036 |
Leaf area |
0.992 |
0.062 |
-0.109 |
Scion fresh weight |
0.868 |
0.487 |
0.099 |
Root fresh weight |
0.93 |
0.364 |
-0.055 |
Nitrogen (leaf) |
-0.435 |
0.88 |
-0.192 |
Nitrogen (root) |
-0.608 |
0.736 |
0.298 |
Phosphorus (leaf) |
0.871 |
-0.226 |
-0.437 |
Phosphorus (root) |
0.588 |
-0.772 |
-0.244 |
Potash (leaf) |
0.884 |
0.416 |
0.211 |
Potash (root |
0.674 |
0.303 |
0.674 |
Calcium (leaf |
0.859 |
-0.51 |
0.053 |
Calcium (root) |
0.567 |
-0.601 |
0.564 |
Magnesium (leaf) |
0.902 |
-0.293 |
-0.316 |
Magnesium (root) |
0.766 |
0.578 |
0.28 |
Iron (leaf) |
-0.972 |
0.227 |
-0.06 |
Iron (root) |
-0.007 |
-0.791 |
0.612 |
Copper (leaf) |
0.307 |
-0.903 |
-0.301 |
Copper (root) |
0.66 |
-0.735 |
0.154 |
Manganese (leaf) |
0.901 |
0.349 |
-0.259 |
Manganese (root) |
0.402 |
-0.887 |
0.228 |
Zinc (leaf) |
0.067 |
-0.787 |
0.614 |
Zinc (root) |
0.594 |
-0.804 |
-0.029 |
Indole-3-acetic acid (leaf) |
0.858 |
0.49 |
-0.153 |
Zeatin riboside (leaf) |
1 |
0.011 |
-0.026 |
Gibberellic acid (leaf |
-0.273 |
0.926 |
0.259 |
Abscisic acid (leaf) |
0.07 |
0.866 |
-0.495 |
Indole-3-acetic acid (root) |
0.907 |
0.369 |
-0.2 |
Zeatin riboside (root) |
0.992 |
0.13 |
-0.009 |
Gibberellic acid (root) |
-0.16 |
-0.524 |
0.837 |
Abscisic acid (root) |
-0.598 |
0.72 |
0.352 |
Leaf water potential |
0.91 |
0.316 |
0.27 |
Hydraulic conductance |
0.985 |
-0.12 |
0.125 |
Photosynthesis rate (Pn) |
0.925 |
-0.326 |
0.194 |
Stomatal conductance (Gs) |
0.996 |
-0.089 |
0.028 |
Intercellular CO2 concentration (Ci) |
0.954 |
0.207 |
0.218 |
Transpiration rate (E) |
0.843 |
0.532 |
0.078 |
Starch (leaf) |
-0.452 |
-0.078 |
-0.889 |
Total measured carbohydrates (leaf) |
-0.61 |
0.785 |
0.109 |
Starch (root) |
-0.374 |
0.889 |
0.265 |
Total measured carbohydrates (root) |
-0.558 |
0.801 |
0.216 |
Eigen value |
31.663 |
19.825 |
5.512 |
Percent of variance (%) |
55.594 |
34.781 |
9.670 |
Correlation analysis for the rootstock induced
morphological and biochemical changes
The correlation among
morphological, physiological, and biochemical traits of ‘Red Fuji’ apple trees
grown onto different rootstocks was shown in (Fig. 6). Plant height, shoot
length, number of nodes, internodal length, number of root tips, number of root
forks, and total root length were positively correlated with leaf water
potential, photosynthesis rate, stomatal conductance, and transpiration rate.
In comparison, these parameters were negatively correlated with leaf N, Fe and
Cu concentrations. Shoot length was negatively correlated with starch contents.
Internodal length, trunk diameter of the scion, number of tips, and total root
length were also negatively correlated with leaf and root starch contents.
Plant height, shoot length, trunk diameter of scion, and intermodal length was
positively correlated with leaf P, K, Mg, Mn, IAA and ZR concentrations.
Fig. 6: Correlations of the morphological and biochemical
indices of ‘Red Fuji’ apple grafted onto different rootstocks.
Abbreviations
PLH= plant
height, IL= internodal length, NN= number of nodes, TSD= trunk scion diameter,
SFW= scion fresh weight, RW= root weight, R:S ratio = root: shoot ratio, TSS=
Total soluble sugars, TCL= total carbohydrate in leaf, TCR= total carbohydrate
in root, IAA = indole-3-acetic acid, ZR = zeatin riboside, GA3 =
gibberellic acid, ABA = abscisic acid, P = phosphorus, N = nitrogen, K =
potassium, Ca = calcium, Mg = magnesium, Fe = iron, Mn = manganese, Zn = Zinc,
WUE= water use efficiency, Pn= net photosynthesis rate, E= transpiration rate,
Ci= inter cellular CO2 concentration, Gs = stomatal conductance,
LWP= leaf water potential and HC = hydraulic conductance
Principal component analysis (PCA) for morphological
parameters and biochemical traits
Factor analysis showed that the eigenvalues (Ei) of the first three components were
all greater than one and represented the main factors (Table 7). Eigenvalues
are the variances of the principal components. Because we conducted our
principal component analysis on the correlation matrix, the variables are
standardized, which means that each variable has a variance of 1, and the total
variance is equal to the number of variables used in the analysis. The sum of
the percent of variance (POV) was 100%, with the amount of information
contained in these three factors representing 100% of the total information.
Representative indices (with higher weight) of component 1 included 27
evaluation indices, with POVs of 55.6%. Representative indices of component 2
were leaf (GA3, ABA and Cu) and root (Mn, Zn, and starch) with POVs
of 34.81%. Representative indices for component 3 were leaf (starch) and root
(GA3), with POVs of 9.64%. The selection was performed based on the
weights of the evaluation indices. Plant height, shoot length and number of
nodes (morphological indicator), the number of root tips and root volume (root
system morphology index), Kleaf
(water status), Pn, Gs, Ci
(photosynthesis indices), leaf K, Mg, Fe and Mn (minerals), Leaf IAA and GA3
and root IAA, ZR (hormones) were selected suitable indices for evaluating the
dwarfing potential of different apple rootstocks.
Discussion
Vigour controlling rootstocks are frequently used in
commercial apple culture cause a decrease in tree crown dimension (Foster et al.
2017). Various studies have suggested that plants grafted onto vigorous
rootstocks have longer shoot lengths, greater trunk diameter of the scion, and
vigorous growth (Gjamovski and Kiprijanovski
2011). However, dwarfing rootstocks alter shoot morphology by the
decrease in shoot length, node number, and length of sylleptic shoots (Costes et al.
2001; Hooijdonk et al. 2010).
In this study, we observed that the ‘Red Fuji’ apple grafted onto ‘M.9’
rootstock induced dwarf size trees, however taller/vigorous rootstock such as
‘Chistock-1’ and ‘Baleng’ rootstocks extended the primary shoot length and
whole tree architecture (Fig. 1 and Table 2).
Root
morphology and architecture of dwarfing rootstocks differ from more vigorous
rootstocks (Kumar et al. 2018). Root morphological characters can be used for
selecting dwarf rootstocks (Luo et al. 2014). In this study, we
found that the root system of ‘Baleng’ rootstock was stronger with greater root
surface area, root volume, projected area, root length, number of tips, and
forks of ‘Red Fuji’ apple trees (Table 3). The difference in root morphological
traits of rootstocks may be attributed to the genetic alterations among
rootstocks (Eissenstat 1991).
A
differential ability to synthesize hormones has been associated with
rootstock-induced dwarfing mechanism (Hooijdonk et al. 2011). Auxin and cytokinin
encourage sprouting of axillary buds and tree vigour. Gibberellin favours
internodal enlargement, whereas ABA stimulates tree ageing (Yu et al.
2012). In the present study, the leaf IAA levels were positively
correlated with tree size, being lowest with ‘M.9’, ‘M.26’, and ‘Chistock-1’
rootstocks; however, the highest was recorded in ‘Baleng’ rootstock (Fig. 2A). Hayat et al.
(2019) reported that ‘Red Fuji’ apple trees grafted onto ‘Baleng’
rootstock were highest in IAA levels. However, trees grafted onto dwarfing
rootstocks presented lower values of IAA. Furthermore, ‘Red Fuji’ apple trees
grafted with ‘M.9’ rootstock showed lower root IAA levels (Fig. 2B), probably
because of lower basipetal IAA transport than vigorous rootstock (Soumelidou et
al. 1994). Cytokinin is produced in the root portion and transported
upwards through the xylem stream (Aloni et al. 2005). ZR levels were
positively correlated with growth parameters. Trees grafted onto ‘M.9’
rootstock were lowest in leaf and root ZR levels, whereas the highest was found
onto ‘M.26’, ‘Chistock-1’, and ‘Baleng’ rootstocks (Fig. 2C and 2D). The
reduction of scion growth in ‘Red Fuji’ apple grafted onto ‘M.9’ rootstock is
due to limited IAA basipetal transport that, in response, decreased
root-derived cytokinin contributed to adequate zeatin shortage in scion potion
and consequently suppressed plant growth (Zhang et al. 2015). Therefore, a
reduced capacity for cytokinin production and movement may be the primary
reasons that restrict tree size.
YUCCA
monooxygenases catalyzing the conversion of indole-3-pyruvate to IAA has a
crucial role in auxin synthesis (Won et al. 2011). The expression
levels of the YUCCA10a gene in leaf
and roots were positively correlated with morphological traits and IAA levels
of apple trees (Fig. 3A and 3B). The low IAA content in dwarfing rootstock
(‘Red Fuji’/‘M.9’) may be attributed to the weaker ability of IAA synthesis in
the root and limited root development. The previous study found that ‘Fuji’
apple grafted onto ‘M.9’ rootstock had lower expression levels of IAA synthesis
gene MdYUCCA10a, possibly induced the
lower level of auxin (Song et al. 2016). According to another report, IPT3 shows a vital role, such as the
regulator for CK synthesis in Arabidopsis
(Takei et
al. 2004; Peng et al. 2008).
In our trial, relative expression of IPT3A
gene and ZR content in leaves and roots were much lower in dwarfing rootstock
(‘Red Fuji’/‘M.9’) compared with more vigorous rootstocks (Fig. 3C and 3D).
This suggests that IPT3A actively
regulates CK synthesis and movements. Reduced expression of IPT3A resulted in reduced ZR synthesis
in the roots of dwarfing rootstock (‘Red Fuji’/‘M.9), which led to the decrease
of the IAA level in the above-ground part (scion) and reduced growth vigour.
Growth
vigour is closely linked with water transport ability and leaf water potential
of plants (Jones et al. 2010). For ‘M.9’ rootstock, which had stronger
size-controlling characteristics, the values of ψleaf and Kleaf
were lesser compared with ‘M.26’, ‘Chistock-1’, and ‘Baleng’ rootstock that had
vigorous/taller characteristics (Fig. 4E and 4F). These findings were
consistent with a previous study (Zhao et al. 2016). Therefore, stronger
dwarfing effects are caused by hydraulic limitations because the lower capacity
of water transport reduced photosynthetic activities, which affect biomass
production (Atkinson et al. 2003; Webster and Wertheim 2003).
The
photosynthetic characteristics of grafted plants are positively correlated with
growth vigour (Sabajeviene et al. 2006; Nawaz et al.
2018). In the current research, plants grafted onto ‘M.9’ rootstock
presented lower photosynthetic rates compared with ‘M.26’, ‘Chistock-1’, and
‘Baleng’ rootstocks (Fig. 4A). Numerous studies have reported that the
photosynthesis values of trees grown onto size-controlling rootstocks were
markedly lower than trees grown on vigorous rootstocks (Fallahi et al. 2001; Gonçalves et al. 2006; Sotiropoulos 2008).
It may be elucidated that size-controlling rootstocks decreased the ability of
water transport, prompting the degree of stomatal opening and CO2 assimilation.
Subsequently, the growth vigour of plants are weakened, and the efficiency of
photosynthesis declines (Brodribb and Feild
2000). Therefore, photosynthesis rates (Pn) may be used as an indicator to access the dwarfing
ability of a rootstock.
Several
studies suggest that vigour of
rootstock has a substantial effect on the uptake of mineral nutrients in apple
trees (Fallahi et al. 2001; Kucukyumuk and Erdal 2011). In our study, ‘Red
Fuji’ apple trees grafted onto ‘M.9’ rootstock was less efficient than ‘M.26’,
‘Chistock-1’, and ‘Baleng’ rootstocks in the absorption of mineral nutrients.
Lower mineral uptake capacity was associated with the root system (total root
length, number of tips, and forks) that can directly affect nutrient uptake
efficiencies.
Rootstocks
influence dynamics of nutrient status may be elucidated as alterations in root
distributions, and root functions that affect mineral uptake ability and
possible variations in stem and root anatomical structures (Zarrouk et al.
2005). The capability of the hydraulic status to supply nutrients
through roots to the leaf is related to anatomical characteristics (Atkinson et al.
1998). The lower hydraulic conductance will also reduce the rate of
nutrient uptake and growth vigour, and this has been suggested as a possible
mechanism of scion control (Higgs and Jones
1991).
Xylem
characteristics, such as the diameter of vessel and density, considerably
influence the growth morphology of scion and rootstocks in grafted fruit plants
(Tombesi et
al. 2011; Chen et al. 2015).
In the current study, leaf anatomical studies showed that ‘Red Fuji’/M-9’ had
significantly lower xylem vessel diameter and xylem vessel density compared
with ‘M-26’, ‘Chistock-1’ and ‘Baleng’ rootstocks (Fig. 5). The lower growth of
‘M.9’ might be linked with anatomical structures. These characteristics play an essential
role in hydraulic status (Hajagos and Végvári
2012; Tombesi et al. 2011).
Apple and peach dwarfing rootstocks had significantly lower hydraulic
conductance compared with semi-dwarfing and taller/stronger rootstocks because
dwarfing rootstocks have smaller and fewer xylem vessels (Atkinson et al.
2003; Basile et al. 2003a).
Lower hydraulic conductance of dwarfing rootstock decreases the stem water
potential, limits stomata opening, and consequently lesser photosynthetic
assimilation (Zorić et al. 2012).
Beakbane
(1956) reported that apple plants grafted onto size-controlling rootstocks have
smaller xylem vessels compared with semi-dwarfing and vigorous rootstocks, and
that might lead to reduced hydraulic conductance with causing of enhanced water
deficits and therefore reduced growth vigour. Besides, Bauerle et al. (2011)
reported that the xylem vessel diameter of apple trees grafted onto dwarfing
B.9 rootstock decreased compared with MM.111 rootstock in response to drought.
The higher accumulation of starch in dwarfing rootstock may be explained by the
accumulation of non-structural carbohydrates in leaves inhibits photosynthetic
activities that affect plant growth (Araya et al. 2006). The previous study
showed that trees grafted onto dwarfing citrus rootstocks accumulated higher
starch concentrations compared with vigorous rootstocks (Mendel and Cohen 1967). We observed a negative correlation among
leaf and root starch contents with morphological traits (including shoot
length, number of root tips). Therefore, these indices may be used for
selecting dwarfing apple rootstocks.
Conclusion
This study demonstrates that morphological,
physiological, and biochemical indices of ‘Red Fuji’ apples were affected by
rootstocks. These indicators can be utilized for the new selection of dwarfing
apple rootstocks. The dwarfing rootstock ‘M-9’ seems to induce lower plant
height based on lower shoot length and number of nodes (tree morphological
index), the number of root tips and root volume (root system morphology index),
Pn, Gs, Ci
and Kleaf (physiological indices), K, Mg, Fe, Mn and starch
(nutrients), IAA, GA3 (hormones). These are suitable indices for
evaluating the dwarfing potential of different rootstocks.
Acknowledgments
This project was funded by the earmarked fund for China
Agriculture Research System (CARS-27), National Natural Science Foundation
(31801810), National Science Foundation (31772262) and grants from the Beijing
Municipal Education Commission (CEFF-PXM2019_014207_000099).
Author Contributions
Faisal Hayat and Chanpeng Qiu conceived and designed the
experiments; Faisal Hayat, Zhou Yanmin and Tian Xue performed the experiments;
Summera Asghar and Faisal Hayat analyzed the data; Xuefeng Xu, Ting Wu,
Xinzhong Zhang and Wang Yi contributed reagents/materials/analysis tools;
Faisal Hayat wrote the paper; Muhammad Azher Nawaz and Zhenhai Han revised the
article.
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