Abstract
Root pruning can promote the transplanting of young green plants, but
the overall impact of pruning on root growth, morphology, and
physiological functions remains unclear. This study integrated
transcriptomics and physiological analyses to elucidate the effects of
root pruning on blueberry growth. Appropriate pruning (CT4)
significantly promoted plant growth, with above-ground biomass and leaf
biomass significantly increasing compared to the control group within
42 days. Photosynthesis temporarily decreased at 7 days but recovered
at 21 and 42 days. Transcriptomics analysis showed that the cellulose
metabolism pathway was rapidly activated and influenced multiple key
genes in the starch metabolism pathway. Importantly, transcription
factors associated with vascular development were also significantly
increased at 7, 21, and 42 days after root pruning, indicating their
role in regulating vascular differentiation. Enhanced aboveground
growth was positively correlated with the expression of
photosynthesis-related genes, and the transport of photosynthetic
products via vascular tissues provided a carbon source for root
development. Thus, root development is closely related to leaf
photosynthesis, and changes in gene expression associated with vascular
tissue development directly influence root development, ultimately
ensuring coordinated growth between aboveground and belowground parts.
These findings provide a theoretical basis for optimizing root pruning
strategies to enhance blueberry growth and yield.
Keywords: root pruning, root growth, transcriptome, vascular tissue
development
1. Introduction
The quality of seedling growth directly influences subsequent growth
and yield, rendering it an essential factor in agricultural production
[[40]1]. Nutrient bowl seedling cultivation is widely recognized as an
effective method for seedling cultivation [[41]2]. Although nutrient
pot nurseries can maintain the relative integrity of the root system,
they also present deficiencies such as reduced capillary roots as well
as coiled, aged, and deformed root systems. Therefore, bare-root
seedling transplantation remains widely practiced [[42]3], with root
pruning being a common technique to facilitate seedling transplantation
and transportation [[43]4]. Root pruning is a common artificial root
disturbance technique used in seedling nurseries, whereby the
regeneration and optimization of the plant root system are stimulated
by subtracting a portion of the seedling’s primary root, thereby
improving plant growth performance and health [[44]5]. Previous studies
on grafted thin-shelled walnut seedlings have shown that an optimal
root-cutting ratio could enhance root length, diameter, and total dry
weight, thereby improving the quality indices of seedlings [[45]6].
This research also explored the effects of various root pruning methods
by examining characteristics including the root surface area, root
length, root volume, number of roots, and root diameter. The results
demonstrated that appropriately proportioned root pruning could improve
the root morphology of the plant, which in turn enhances the quality
indicators of the seedlings [[46]7]. It has been reported that properly
proportioned root pruning improved water use efficiency in corn plants
[[47]8]. Root pruning is an effective method for improving plant
photosynthetic traits, increasing fruit yield [[48]9,[49]10,[50]11],
and promoting plant growth [[51]12].
Root pruning, as an agricultural management practice, exerts a
significant effect on plant growth and development, a process that
involves complex physiological phenomena and morphological changes
[[52]13,[53]14]. First, root pruning stimulates a range of
physiological responses in plants, most notably changes in the
phytohormone content. Interestingly, it has been reported that root
pruning could induce significant changes in the contents of
phytohormones, such as increases in the contents of indole-3-acetic
acid (IAA), jasmonic acid (JA), and abscisic acid (ABA) in roots, which
are usually involved in plant growth and defense responses [[54]15].
Moreover, under stress or disturbance conditions, such as root pruning,
the plant antioxidative enzyme system is activated to scavenge the
excessive reactive oxygen species (ROS) [[55]16]. Overall, changes in
the contents and activities of phytohormones and enzymes contribute to
the recovery of the pruned roots or the initiation of lateral roots
following pruning [[56]17,[57]18].
In addition, root pruning affects the lignin and cellulose content of
the root system. Lignin, an important secondary metabolite, enhances
the mechanical strength of the cell wall and improves the stability of
the root system [[58]19]. After pruning, the root system increases
lignin synthesis in response to wounds and possible pathogen invasion,
thus forming a protective barrier around the wound [[59]20]. At the
same time, cellulose synthesis may be regulated to maintain cell wall
structure and function. Morphologically, the significant enlargement of
vascular tissue that root pruning induces is the result of the high
expression of vascular-related genes around the edge of the root
incision [[60]21]. Vascular tissue is the main structure responsible
for material transport in plants, including xylem and phloem. After
pruning, vascular tissues undergo a series of adaptations to accelerate
wound healing and root recovery. In this respect, it has been shown
that xylem conduits may become more developed to improve the efficiency
of water and inorganic salt transport [[61]22,[62]23,[63]24], while
phloem sieve tubes may enhance their ability to transport the products
of photosynthesis to provide an adequate energy and material base for
root growth [[64]25,[65]26]. Furthermore, root pruning exerts
substantial effects on the development of vascular tissues. During the
post-pruning recovery process, vascular tissues undergo a series of
complex processes such as cell division, differentiation, and
programmed death to form new ducts and sieve tube systems. These newly
formed vascular tissues not only demonstrate refined transport
efficiency but also exhibit enhanced adaptability to environmental
changes, contributing to improved plant resistance and productivity.
Although root pruning techniques are widely applied in crop cultivation
management, current research still faces two major limitations: first,
the physiological response mechanisms triggered by pruning (such as
dynamic changes in photosynthesis and adjustments in carbon-nitrogen
allocation) and their association with molecular-level events (such as
vascular tissue regeneration) remain fragmented and lack systematic
integration. Second, the specific differences between woody and
herbaceous plants in root regeneration remain unclear, particularly
whether shallow-rooted woody plants (such as blueberries) possess
unique molecular pathways and structural reconstruction patterns for
regeneration, which remains undecided. These two limitations constrain
the precise application of root pruning techniques in perennial woody
economic crops.
Seedling nurseries are important for the development of the blueberry
industry. As a woody plant characterized by fibrous, shallow, and
relatively weak root system, blueberry plants have high requirements
for growing soil media, and the substrate potting method has gradually
become a prevalent cultivation trend [[66]27]. However, with the growth
of potted blueberries, the root system growth is constrained by the
container, leading to significant entanglement against the pot walls
and a resultant insufficient supply of plant nutrients. Therefore,
practical management necessitates providing the root system with more
space to grow (e.g., change to larger-size containers) or employing
appropriate techniques to remove redundant roots (e.g., root pruning)
to promote root regeneration, which in turn affects plant growth.
Root pruning is an important management measure in blueberry nurseries;
therefore, analyzing the molecular mechanisms of blueberry root pruning
can improve blueberry cultivation efficiency. The present study aims to
investigate the regulatory mechanism of root pruning regarding
blueberry root development at the molecular level, which holds great
scientific significance for revealing the molecular basis by which
partial primary root removal promotes the development of lateral roots
and provides important scientific data for the propagation of woody
plants.
The selection of Vaccinium corymbosum as a model system is based on its
unique biological constraints, economic importance, and unresolved
molecular mechanisms. First, unlike deep-rooted woody perennials,
blueberries have shallow, fibrous root systems with extremely low root
hair development. This morphological specialization makes them highly
susceptible to root-restricted stress in container cultivation, thereby
enhancing phenotypic responses to pruning interventions [[67]28].
Second, as the world’s most valuable berry crop [[68]29], China’s
rapidly developing blueberry industry faces a 30% yield gap due to
transplant losses, a gap that could be addressed through optimized root
management. Finally, while root pruning can enhance yields in Rosaceae
crops, little research has been conducted on transcription regulatory
factors associated with vascular bundle regeneration in Ericaceae
plants, leaving a critical knowledge gap for breeding programs aimed at
developing stress-tolerant root systems.
2. Materials and Methods
2.1. Plant Material and Experimental Design
The experimental materials were selected from 6-month-old cuttings of
the blueberry variety ‘Eureka’ provided by Dalian Senmao Modern
Agriculture Co., Ltd. in Dalian, China. The trial was conducted in the
greenhouse of Dalian University Cooperative Experimental Base from
April to June 2023. In April 2023, the average daily temperature ranged
from 8 °C to 16 °C, with an average relative humidity of 68%. Under
clear weather conditions, the photosynthetically active radiation (PAR)
at noon can reach 12,000 μmol/(m^2·s). The experimental photoperiod was
14 h/10 h (light duration/dark period). A one-way randomized block
design was applied, setting no root pruning as the control group and
six treatment groups involving the pruning of 20%, 40%, 50%, 60%, 80%,
and 100% of the root system, with a total of seven experimental groups
and five experimental seedlings in each group. In April 2023, 35
blueberry seedlings cultivated in the self-developed root culture
observation device, exhibiting good growth conditions and having been
cultivated in a custom-designed root culture observation device (where
root growth had reached the glass plate edges, indicating restriction),
were selected for root pruning treatments (pruning only once) centered
on the root node, at both sides and the bottom of the root clipping
treatments to maintain the root width of the root in equal proportions
to reduce the root system by 20% (CT2), 40% (CT4), 50% (CT5), 60%
(CT6), and 80% (CT8). The 100% pruning treatment involved the retention
of only the primary root 1–2 cm below the root node. Calculate the area
to be cut according to the schematic diagram in [69]Figure S3 of
Supplementary Information 2. Use an ethanol-disinfected blade to cut at
a 90° angle at the root cutting site.
The custom-designed root culture observation device consisted of a 30
cm × 30 cm glass plate clamped on both sides, separated by a 1 cm glass
sealing strip in the middle, and three 5 cm dovetail clamps were used
on both sides to clamp the upper, middle, and lower parts,
respectively, with aluminum-plated reflective film on the outer cover
to block out the light. The schematic diagram is shown in [70]Figure S2
in Supplementary Information 2. This study utilized a growing medium
composed of perlite, peat moss, and coconut coir mixed at a 1:1:1
volume ratio, which was subjected to high-temperature sterilization
treatment (121 °C for 30 min) to eliminate pathogens and weed seeds.
Its physical and chemical properties are as follows: total porosity of
85–90%, water-holding capacity of 60–75%, combining good aeration and
water retention, pH of 5.5–6.5, slightly acidic, organic matter content
of 30–40%, and electrical conductivity of 0.3–0.5 mS/cm (unfertilized).
This formulation aligns with standard blueberry cultivation practices,
providing optimal aeration, acidity, and water retention for fibrous
root development. The nutrient solution was watered twice a day during
the experimental period, based on the Hoagland and Arnon nutrient
solution [[71]30], and supplemented with trace elements from other
common formulas within a pH range of 4.0–4.5.
2.2. Individual Morphometry
At 7, 21, and 42 days after the experimental treatments, the glass
panel on one side of the root growth observation device was opened, and
a scanner (SinocrystalScanMaker 9800XL, Shanghai, China) was used to
scan and record the root growth status of the seedlings in each group
of experimental seedlings, with three blueberry plants selected from
each group. The height and basal stem of blueberry plants were measured
using a steel tape measure (accuracy 0.1 cm) and vernier caliper
(accuracy 0.01 mm) at 7 d, 21 d, and 42 d after root pruning treatment,
respectively. The number of leaves on each plant were recorded,
selecting three biological replicates for each group. Leaves were
scanned with a scanner at the end of the treatment and, subsequently,
the average leaf area was calculated using Adobe Photoshop 2019. The
calculation formula was as follows [[72]31]:
[MATH: Leaf area(cm2)=Number of leaves×Average leaf area :MATH]
Forty-two days after the end of the treatment, three blueberry plants
exhibiting uniform growth were randomly selected from each treatment
group. The leaves and stems were rinsed, dried, and weighed at 0.5 g
each using a balance with a precision of 0.001 g. The root system of
each blueberry plant was washed with distilled water, wiped dry, and
weighed using a balance with a precision of 0.0001 g to obtain 0.2 g,
with three replicates. Subsequently, the weighed blueberry leaves,
stems, and root systems were labeled and put into the oven for
inactivation at 110 °C for half an hour and then baked at 75 °C until
they reached a constant weight. The dry weight was then recorded
separately. The calculation formula was as follows [[73]32]:
[MATH: Water Content(%)=(Fresh weight−Dry weight)/Dry weight×100 :MATH]
2.3. Determination of Photosynthetic and Chlorophyll Fluorescence Parameters
Photosynthetic parameters including the photosynthetic rate (Pn),
transpiration rate (Tr), stomatal conductance (Gs), and substomatal
cavity CO[2] concentration (Ci) of the same functional leaves of
blueberry plants in the root pruning group and the control group were
measured using the Li-6400 XT Portable Photosynthesis (Beijing ecotek
Company, Beijing, China) Instrument between 9:00 a.m. and 11:00 a.m. at
7, 21, and 42 d after the experimental treatments. Photosynthesis
parameters were measured on three biologically independent plants in
each treatment group. For each plant, three mature leaves (located at
the third to fifth nodes from the top) were selected as technical
replicates, and measurements were taken between 9:00 and 11:00 a.m. on
a clear day.
The chlorophyll fluorescence parameters, including maximal
photochemical efficiency (F[v]/F[m]), photosystem II (PSII), and
nonphotochemical quenching (NPQ), were assessed using the Li-6400 XT
Portable Photosynthesis Instrument at the same time as the
photosynthetic measurements.
2.4. RNA Extraction, cDNA Library Construction, and Transcriptome Sequencing
RNA was extracted from roots and leaves at different developmental
stages from the pruning group and the control group. All freeze-dried
samples were ground into powder in liquid nitrogen, and 50 mg of powder
was used for total RNA extraction using an RNA extraction kit (Beijing
Adelaide Biotechnology Co., Ltd., Beijing, China). The RNA
concentration, RIN value, 28S/18S ratio, and fragment size were
measured using an Agilent 2100 Bioanalyzer (Beijing, China) to assess
RNA integrity. RNA purity and concentration were determined using a
NanoDrop™ UV-visible spectrophotometer (Thermo Fisher Scientific Inc.,
Shanghai, China) based on the OD260/280 ratio.
The DNBSEQ platform was used for library construction and quality
control. After the samples passed the test, the library was constructed
according to the following steps: enrich mRNA with magnetic beads with
Oligo (dT) and add the fragmentation buffer to interrupt mRNA. The
first cDNA strand was synthesized with six-base random hexamers using
mRNA as a template, and then the second cDNA strand was synthesized by
adding buffer, deoxynucleotide mix (dNTPs), Ribonuclease H (RNaseH),
and DNA polymerase I. Then, double-stranded cDNA was purified using a
kit (Thermos Fisher Scientific Inc., Shanghai, China). The purified
double-stranded cDNA was terminal-repaired, and a tail was added with a
sequencing joint connected. Finally, PCR amplification was performed to
construct the sequencing library. After the library was constructed,
the insert range of the library was examined using an Agilent 2100
Bioanalyzer. The ABI StepOnePlus Real-Time PCR System was used to
quantify the library concentrations. After passing the quality
inspection, sequencing was performed using an Illumina platform
sequencer (Beijing, China).
2.5. Functional Analysis of Differentially Expressed Genes
The predicted new genes were functionally annotated using the NR, GO,
and KEGG databases to obtain detailed descriptions of the new genes.
Cleaned read sequences were aligned to the reference genome Vaccinium
darrowii v1.2 using HISAT2 v2.2.1. Transcriptome reconstruction was
performed for each sample using StringTie. Subsequently, CPC was used
to predict the protein-coding potential of novel transcripts, and novel
transcripts identified as potential protein-coding transcripts were
added to the reference gene sequence to generate a comprehensive
reference sequence, which was then used for subsequent analyses.
Differential expression analysis was performed using DESeq2, with a
threshold of |log2Fold Change| > 1 and q-value < 0.001 to identify
differentially expressed genes. Finally, GETORF was used to detect the
open reading frames (ORFs) of each differentially expressed gene (DEG),
and the ORFs were compared with transcription factor protein domains
(from PlantTFDB) using hmmsearch. Then, the transcription factor
encoding capability characteristics of DEGs were analyzed based on the
transcription factor family characteristics described in PlantfDB.
2.6. Identification of Transcription Factors Related to Vascular Development
To systematically analyze the regulatory mechanisms underlying root
pruning-induced vascular tissue development, this study screened and
validated key TFs related to vascular tissue differentiation in
blueberry based on public databases and homology comparison strategies.
The specific process was as follows: 1. Transcription factor data
sources: reported vascular tissue development-related transcription
factor families were downloaded as protein sequences from NCBI RefSeq
(Visited from 15 January 2025 to 19 March 2025;
[74]https://www.ncbi.nlm.nih.gov/) and Phytozome v13 (Visited from 10
January 2025 to 3 March 2025; [75]https://phytozome-next.jgi.doe.gov/)
(e.g., NAC, AP2/ERF, MYB, HD-ZIP) for model plants such as Arabidopsis
thaliana and Populus trichocarpa. The predicted transcription factor
sequences were extracted by integrating the gene annotation file of the
blueberry (Vaccinium corymbosum) reference genome Vaccinium darrowii
clone NJ8810/NJ8807 v1.2 genome sequence. 2. Homology comparison and
candidate gene screening involved the local alignment of the blueberry
genome against model plant development-related TFs using BLAST+ v2.13.0
as query sequences, based on the following thresholds: E-value ≤ 1 ×
10^−5, coverage ≥ 60%, and sequence identity ≥ 40%. A Neighbor-Joining
phylogenetic tree was constructed using MEGA 11. The known functional
TFs of Arabidopsis thaliana and Populus trichocarpa were used as
references to cluster the blueberry candidate genes and infer their