A rapid and highly efficient sorghum transformation strategy using GRF4-GIF1/ternary vector system.

Sorghum is an important crop for food, forage, wine and biofuel production. To enhance its transformation efficiency without negative developmental by-effects, we investigated the impact of GRF4-GIF1 chimaera and GRF5 on sorghum transformation. Both GRF4-GIF1 and GRF5 effectively improved the transformation efficiency of sorghum and accelerated the transformation process of sorghum to less than 2 months which was not observed when using BBM-WUS. As agrobacterium  effectors increase the ability of T-DNA transfer into plant cells, we checked whether ternary vector system can additively enhance sorghum transformation. The combination of GRF4-GIF1 with helper plasmid pVS1-VIR2 achieved the highest transformation efficiency, reaching 38.28%, which is 7.71-fold of the original method. Compared with BBM-WUS, overexpressing GRF4-GIF1 caused no noticeable growth defects in sorghum. We further developed a sorghum CRISPR/Cas9 gene-editing tool based on this GRF4-GIF1/ternary vector system, which achieved an average gene mutation efficiency of 41.36%, and null mutants were created in the T0 generation.

Leaf growth - complex regulation of a seemingly simple process.

Understanding the underlying mechanisms of plant development is crucial to successfully steer or manipulate plant growth in a targeted manner. Leaves, the primary sites of photosynthesis, are vital organs for many plant species, and leaf growth is controlled by a tight temporal and spatial regulatory network. In this review, we focus on the genetic networks governing leaf cell proliferation, one major contributor to final leaf size. First, we provide an overview of six regulator families of leaf growth in Arabidopsis: DA1, PEAPODs, KLU, GRFs, the SWI/SNF complexes, and DELLAs, together with their surrounding genetic networks. Next, we discuss their evolutionary conservation to highlight similarities and differences among species, because knowledge transfer between species remains a big challenge. Finally, we focus on the increase in knowledge of the interconnectedness between these genetic pathways, the function of the cell cycle machinery as their central convergence point, and other internal and environmental cues.

Use of GRF-GIF chimeras and a ternary vector system to improve maize (Zea mays L.) transformation frequency.

Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium-mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene-editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1-VIR2 virulence helper plasmid to improve T-DNA delivery, and/or expressing a fusion protein between a GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1-VIR2 helper vector with no effect on event quality regarding T-DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5- to 6.5-fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR-/Cas9-mediated gene editing. Our results demonstrate how a GRF-GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene-editing applications and molecular biology studies in maize.

Genome-wide molecular evolution analysis of the GRF and GIF gene families in Plantae (Archaeplastida).

BACKGROUND: Plant growth-regulating factors (GRFs) and GRF-interacting factors (GIFs) interact with each other and collectively have important regulatory roles in plant growth, development, and stress responses. Therefore, it is of great significance to explore the systematic evolution of GRF and GIF gene families. However, our knowledge and understanding of the role of GRF and GIF genes during plant evolution has been fragmentary. RESULTS: In this study, a large number of genomic and transcriptomic datasets of algae, mosses, ferns, gymnosperms and angiosperms were used to systematically analyze the evolution of GRF and GIF genes during the evolution of plants. The results showed that GRF gene first appeared in the charophyte Klebsormidium nitens, whereas the GIF genes originated relatively early, and these two gene families were mainly expanded by segmental duplication events after plant terrestrialization. During the process of evolution, the protein sequences and functions of GRF and GIF family genes are relatively conservative. As cooperative partner, GRF and GIF genes contain the similar types of cis-acting elements in their promoter regions, which enables them to have similar transcriptional response patterns, and both show higher levels of expression in reproductive organs and tissues and organs with strong capacity for cell division. Based on protein-protein interaction analysis and verification, we found that the GRF-GIF protein partnership began to be established in pteridophytes and is highly conserved across different terrestrial plants. CONCLUSIONS: These results provide a foundation for further exploration of the molecular evolution and biological functions of GRF and GIF genes.

Genome-wide identification of the GRF family in sweet orange (Citrus sinensis) and functional analysis of the CsGRF04 in response to multiple abiotic stresses.

BACKGROUND: Citrus is one of the most valuable fruits worldwide and an economic pillar industry in southern China. Nevertheless, it frequently suffers from undesirable environmental stresses during the growth cycle, which severely restricts the growth, development and yield of citrus. In plants, the growth-regulating factor (GRF) family of transcription factors (TF) is extensively distributed and plays an vital part in plant growth and development, hormone response, as well as stress adaptation. However, the systematic identification and functional analysis of GRF TFs in citrus have not been reported. RESULTS: Here, a genome-wide identification of GRF TFs was performed in Citrus sinensis, 9 members of CsGRFs were systematically identified and discovered to be scattered throughout 5 chromosomes. Subsequently, physical and chemical properties, phylogenetic relationships, structural characteristics, gene duplication events, collinearity and cis-elements of promoter were elaborately analyzed. In particular, the expression patterns of the CsGRF genes in response to multiple phytohormone and abiotic stress treatments were investigated. Predicated on this result, CsGRF04, which exhibited the most differential expression pattern under multiple phytohormone and abiotic stress treatments was screened out. Virus-induced gene silencing (VIGS) technology was utilized to obtain gene silenced plants for CsGRF04 successfully. After the three stress treatments of high salinity, low temperature and drought, the CsGRF04-VIGS lines showed significantly reduced resistance to high salinity and low temperature stresses, but extremely increased resistance to drought stress. CONCLUSIONS: Taken together, our findings systematically analyzed the genomic characterization of GRF family in Citrus sinensis, and excavated a CsGRF04 with potential functions under multiple abiotic stresses. Our study lay a foundation for further study on the function of CsGRFs in abiotic stress and hormone signaling response.

miR396b/GRF6 module contributes to salt tolerance in rice.

Salinity, as one of the most challenging environmental factors restraining crop growth and yield, poses a severe threat to global food security. To address the rising food demand, it is urgent to develop crop varieties with enhanced yield and greater salt tolerance by delving into genes associated with salt tolerance and high-yield traits. MiR396b/GRF6 module has previously been demonstrated to increase rice yield by shaping the inflorescence architecture. In this study, we revealed that miR396b/GRF6 module can significantly improve salt tolerance of rice. In comparison with the wild type, the survival rate of MIM396 and OE-GRF6 transgenic lines increased by 48.0% and 74.4%, respectively. Concurrent with the increased salt tolerance, the transgenic plants exhibited reduced H2 O2 accumulation and elevated activities of ROS-scavenging enzymes (CAT, SOD and POD). Furthermore, we identified ZNF9, a negative regulator of rice salt tolerance, as directly binding to the promoter of miR396b to modulate the expression of miR396b/GRF6. Combined transcriptome and ChIP-seq analysis showed that MYB3R serves as the downstream target of miR396b/GRF6 in response to salt tolerance, and overexpression of MYB3R significantly enhanced salt tolerance. In conclusion, this study elucidated the potential mechanism underlying the response of the miR396b/GRF6 network to salt stress in rice. These findings offer a valuable genetic resource for the molecular breeding of high-yield rice varieties endowed with stronger salt tolerance.

Molecular Mechanisms Regulating GRF Activity in Plant Growth, Development, and Environmental Responses.

Plants rely on complex regulatory mechanisms to ensure proper growth and development. As sessile organisms, these mechanisms must be flexible enough to adapt to changes in the environment. The GROWTH-REGULATING FACTORs (GRFs) are plant-specific transcription factors that act as a central hub controlling plant growth and development, offering promising biotechnological applications to enhance plant performance. Here, we analyze the complex molecular mechanisms that regulate GRF activity, and how their natural and synthetic variants can impact on plant growth and development. We describe the biological roles of the GRFs and examine how they regulate gene expression and contribute to the control of organ growth and the plant's response to a changing environment. This review focuses on the premise that unlocking their full biotechnological potential requires a thorough understanding of the various regulatory layers governing GRF activity, the functional divergence among GRF family members and the gene networks that they regulate.

General Regulatory Factor7 regulates innate immune signalling to enhance Verticillium wilt resistance in cotton.

Sessile growing plants are always vulnerable to microbial pathogen attacks throughout their lives. To fend off pathogen invasion, plants have evolved a sophisticated innate immune system that consists of cell surface receptors and intracellular receptors. Somatic embryogenesis receptor kinases (SERKs) belong to a small group of leucine-rich repeat receptor-like kinases (LRR-RLKs) that function as co-receptors regulating diverse physiological processes. GENRAL REGULATORY FACTOR (GRF) proteins play an important role in physiological signalling transduction. However, the function of GRF proteins in plant innate immune signalling remains elusive. Here, we identified a GRF gene, GauGRF7, that is expressed both constitutively and in response to fungal pathogen infection. Intriguingly, silencing of GRF7 compromised plant innate immunity, resulting in susceptibility to Verticillium dahliae infection. Both transgenic GauGRF7 cotton and transgenic GauGRF7 Arabidopsis lines enhanced the innate immune response to V. dahliae infection, leading to high expression of two helper NLRs (hNLR) genes (ADR1 and NRG1) and pathogenesis-related genes, and increased ROS production and salicylic acid level. Moreover, GauGRF7 interacted with GhSERK1, which positively regulated GRF7-mediated innate immune response in cotton and Arabidopsis. Our findings revealed the molecular mechanism of the GRF protein in plant immune signaling and offer potential opportunities for improving plant resistance to V. dahliae infection.

Impact of PCA Pre-Normalization Methods on Ground Reaction Force Estimation Accuracy.

Ground reaction force (GRF) components can be estimated using insole pressure sensors. Principal component analysis in conjunction with machine learning (PCA-ML) methods are widely used for this task. PCA reduces dimensionality and requires pre-normalization. In this paper, we evaluated the impact of twelve pre-normalization methods using three PCA-ML methods on the accuracy of GRF component estimation. Accuracy was assessed using laboratory data from gold-standard force plate measurements. Data were collected from nine subjects during slow- and normal-speed walking activities. We tested the ANN (artificial neural network) and LS (least square) methods while also exploring support vector regression (SVR), a method not previously examined in the literature, to the best of our knowledge. In the context of our work, our results suggest that the same normalization method can produce the worst or the best accuracy results, depending on the ML method. For example, the body weight normalization method yields good results for PCA-ANN but the worst performance for PCA-SVR. For PCA-ANN and PCA-LS, the vector standardization normalization method is recommended. For PCA-SVR, the mean method is recommended. The final message is not to define a normalization method a priori independently of the ML method.

Genome-wide identification of GRF transcription factors and their expression profile in stem meristem of broomcorn millet (Panicum miliaceum L.).

To understand the genome-wide information of the GRF family genes in broomcorn millet and their expression profile in the vegetative meristems, bioinformatic methods and transcriptome sequencing were used to analyze the characteristics, physical and chemical properties, phylogenetic relationship, chromosome distribution, gene structure, cis-acting elements and expression profile in stem meristem for the GRF family members. The results showed that the GRF gene family of millet contains 21 members, and the PmGRF gene is unevenly distributed on 12 chromosomes. The lengths of PmGRF proteins vary from 224 to 618 amino acids, and the isoelectric points are between 4.93-9.69. Each member of the family has 1-4 introns and 2-5 exons. The protein PmGRF13 is localized in both the nucleus and chloroplast, and the rest PmGRF proteins are located in the nucleus. Phylogenetic analysis showed that the 21 GRF genes were divided into 4 subfamilies (A,B,C and D) in broomcorn millet. The analysis of cis-acting elements showed that there were many cis-acting elements involved in light response, hormone response, drought induction, low temperature response and other environmental stress responses in the 2000 bp sequence upstream of the GRF genes. Transcriptome sequencing and qRT-PCR analyses showed that the expression levels of PmGRF3 and PmGRF12 in the dwarf variety Zhang778 were significantly higher than those of the tall variety Longmi12 in the internode and node meristems at the jointing stage, while the expression patterns of PmGRF4, PmGRF16 and PmGRF21 were reverse. In addition, the expression levels of PmGRF2 and PmGRF5 in the internode of Zhang778 were significantly higher than Longmi12. The other GRF genes were not or insignificantly expressed. These results indicated that seven genes, PmGRF2, PmGRF3, PmGRF4, PmGRF5, PmGRF12, PmGRF16 and PmGRF21, were related to the formation of plant height in broomcorn millet.

Whole-genome identification and expression profiling of growth-regulating factor (GRF) and GRF-interacting factor (GIF) gene families in Panax ginseng.

BACKGROUND: Panax ginseng is a perennial herb and one of the most widely used traditional medicines in China. During its long growth period, it is affected by various environmental factors. Past studies have shown that growth-regulating factors (GRFs) and GRF-interacting factors (GIFs) are involved in regulating plant growth and development, responding to environmental stress, and responding to the induction of exogenous hormones. However, GRF and GIF transcription factors in ginseng have not been reported. RESULTS: In this study, 20 GRF gene members of ginseng were systematically identified and found to be distributed on 13 chromosomes. The ginseng GIF gene family has only ten members, which are distributed on ten chromosomes. Phylogenetic analysis divided these PgGRFs into six clades and PgGIFs into two clades. In total, 18 of the 20 PgGRFs and eight of the ten PgGIFs are segmental duplications. Most PgGRF and PgGIF gene promoters contain some hormone- and stress- related cis-regulatory elements. Based on the available public RNA-Seq data, the expression patterns of PgGRF and PgGIF genes were analysed from 14 different tissues. The responses of the PgGRF gene to different hormones (6-BA, ABA, GA3, IAA) and abiotic stresses (cold, heat, drought, and salt) were studied. The expression of the PgGRF gene was significantly upregulated under GA3 induction and three weeks of heat treatment. The expression level of the PgGIF gene changed only slightly after one week of heat treatment. CONCLUSIONS: The results of this study may be helpful for further study of the function of PgGRF and PgGIF genes and lay a foundation for further study of their role in the growth and development of Panax ginseng.

GRF-GIF duo and GRF-GIF-BBM: novel transformation methodologies for enhancing regeneration efficiency of genome-edited recalcitrant crops.

MAIN CONCLUSION: This review describes the potential use of two novel transformation methodologies, GRF-GIF and GRF-GIF-BBM, for improving the regeneration efficiency of genome-edited recalcitrant plants.

GRF-GIF chimeric proteins enhance in vitro regeneration and Agrobacterium-mediated transformation efficiencies of lettuce (Lactuca spp.).

KEY MESSAGE: GRF-GIF chimeric proteins from multiple source species enhance in vitro regeneration in both wild and cultivated lettuce. In addition, they enhance regeneration in multiple types of lettuce including butterheads, romaines, and crispheads. The ability of plants to regenerate in vitro has been exploited for use in tissue culture systems for plant propagation, plant transformation, and genome editing. The success of in vitro regeneration is often genotype dependent and continues to be a bottleneck for Agrobacterium-mediated transformation and its deployment for improvement of some crop species. Manipulation of transcription factors that play key roles in plant development such as BABY BOOM, WUSCHEL, and GROWTH-REGULATING FACTORs (GRFs) has improved regeneration and transformation efficiencies in several plant species. Here, we compare the efficacy of GRF-GIF gene fusions from multiple species to boost regeneration efficiency and shooting frequency in four genotypes of wild and cultivated lettuce (Lactuca spp. L.). In addition, we show that GRF-GIFs with mutated miRNA 396 binding sites increase regeneration efficiency and shooting frequency when compared to controls. We also present a co-transformation strategy for increased transformation efficiency and recovery of transgenic plants harboring a gene of interest. This strategy will enhance the recovery of transgenic plants of other lettuce genotypes and likely other crops in the Compositae family.

Hydrogen sulfide improves salt tolerance through persulfidation of PMA1 in Arabidopsis.

KEY MESSAGE: A new interaction was found between PMA1 and GRF4. H2S promotes the interaction through persulfidated Cys446 of PMA1. H2S activates PMA1 to maintain K+/Na+ homeostasis through persulfidation under salt stress. Plasma membrane H+-ATPase (PMA) is a transmembrane transporter responsible for pumping protons, and its contribution to salt resistance is indispensable in plants. Hydrogen sulfide (H2S), a small signaling gas molecule, plays the important roles in facilitating adaptation of plants to salt stress. However, how H2S regulates PMA activity remains largely unclear. Here, we show a possible original mechanism for H2S to regulate PMA activity. PMA1, a predominant member in the PMA family of Arabidopsis, has a non-conservative persulfidated cysteine (Cys) residue (Cys446), which is exposed on the surface of PMA1 and located in cation transporter/ATPase domain. A new interaction of PMA1 and GENERAL REGULATORY FACTOR 4 (GRF4, belongs to the 14-3-3 protein family) was found by chemical crosslinking coupled with mass spectrometry (CXMS) in vivo. H2S-mediated persulfidation promoted the binding of PMA1 to GRF4. Further studies showed that H2S enhanced instantaneous H+ efflux and maintained K+/Na+ homeostasis under salt stress. In light of these findings, we suggest that H2S promotes the binding of PMA1 to GRF4 through persulfidation, and then activating PMA, thus improving the salt tolerance of Arabidopsis.

Normative spatiotemporal and ground reaction force data for female and male sprinting.

This study aimed to demonstrate the differences in spatiotemporal and ground reaction force (GRF) variables during overground sprinting between performance levels for female and male sprinters with providing normative data during the entire acceleration phase. Forty-four female and 102 male sprinters performed 60-m sprints, during which the spatiotemporal and GRF variables were obtained using a long force platform system. Female and male sprinters were each allocated into four groups based on their maximal speed (7.5-9.5 m/s and 8.5-10.5 m/s, respectively) with 0.5 m/s intervals, and average values for 50-m distance were calculated. Using the GRF data, normative data for four groups of female and male sprinters were successfully obtained. For female sprinters using average values of all steps, there were differences between performance levels for step frequency (SF) and support time (ST), all impulses, and all mean forces. For male sprinters using average values of all steps, there were differences between performance levels for SF, ST and flight time, all impulses except for braking impulse, and all of the mean forces. The normative data indicate that most of the spatiotemporal and GRF variables may be changed, particularly increasing SF and propulsive force, when sprint performance is improved.

Genome-Wide Identification and Characterization of the GRF Gene Family in Melastoma dodecandrum.

Growth-regulating factor (GRF) is a kind of transcription factor unique to plants, playing an important role in the flowering regulation, growth, and development of plants. Melastoma dodecandrum is an important member of Melastomataceae, with ornamental, medicinal, and edible benefits. The identification of the GRF gene family in M. dodecandrum can help to improve their character of flavor and continuous flowering. The members of the GRF gene family were identified from the M. dodecandrum genome, and their bioinformatics, selective pressure, and expression patterns were analyzed. The results showed that there were 20 GRF genes in M. dodecandrum. Phylogenetic analysis showed that the 71 GRF genes from M. dodecandrum, Arabidopsis thaliana, Camellia sinensis, and Oryza sativa can be divided into three clades and six subclades. The 20 GRF genes of M. dodecandrum were distributed in twelve chromosomes and one contig. Furthermore, the gene structure and motif analysis showed that the intron and motif within each clade were very similar, but there were great differences among different clades. The promoter contained cis-acting elements related to hormone induction, stress, and growth and development. Different transcriptomic expression of MdGRFs indicated that MdGRFs may be involved in regulating the growth and development of M. dodecandrum. The results laid a foundation for further study on the function and molecular mechanism of the M. dodecandrum GRF gene family.

Genome-Wide Analysis of Flax (Linum usitatissimum L.) Growth-Regulating Factor (GRF) Transcription Factors.

Flax is an important cash crop globally with a variety of commercial uses. It has been widely used for fiber, oil, nutrition, feed and in composite materials. Growth regulatory factor (GRF) is a transcription factor family unique to plants, and is involved in regulating many processes of growth and development. Bioinformatics analysis of the GRF family in flax predicted 17 LuGRF genes, which all contained the characteristic QLQ and WRC domains. Equally, 15 of 17 LuGRFs (88%) are predicted to be regulated by lus-miR396 miRNA. Phylogenetic analysis of GRFs from flax and several other well-characterized species defined five clades; LuGRF genes were found in four clades. Most LuGRF gene promoters contained cis-regulatory elements known to be responsive to hormones and stress. The chromosomal locations and collinearity of LuGRF genes were also analyzed. The three-dimensional structure of LuGRF proteins was predicted using homology modeling. The transcript expression data indicated that most LuGRF family members were highly expressed in flax fruit and embryos, whereas LuGRF3, LuGRF12 and LuGRF16 were enriched in response to salt stress. Real-time quantitative fluorescent PCR (qRT-PCR) showed that both LuGRF1 and LuGRF11 were up-regulated under ABA and MeJA stimuli, indicating that these genes were involved in defense. LuGRF1 was demonstrated to be localized to the nucleus as expected for a transcription factor. These results provide a basis for further exploration of the molecular mechanism of LuGRF gene function and obtaining improved flax breeding lines.

Genome-Wide Identification and Evolution of the GRF Gene Family and Functional Characterization of PbGRF18 in Pear.

Proteins encoded by the G-box regulating factor (GRF, also called 14-3-3) gene family are involved in protein-protein interactions and mediate signaling transduction, which play important roles in plant growth, development, and stress responses. However, there were no detailed investigations of the GRF gene family in pear at present. In this study, we identified 25 GRF family members in the pear genome. Based on a phylogenetic analysis, the 25 GRF genes were clustered into two groups; the ε group and the non-ε group. Analyses of the exon-intron structures and motifs showed that the gene structures were conserved within each of the ε and non-ε groups. Gene duplication analysis indicated that most of the PbGRF gene expansion that occurred in both groups was due to WGD/segmental duplication. Phosphorylation sites analysis showed that the main phosphorylation sites of PbGRF proteins were serine residues. For gene expression, five PbGRF genes (PbGRF7, PbGRF11, PbGRF16, PbGRF21, and PbGRF23) were highly expressed in fruits, and PbGRF18 was highly expressed in all tissues. Further analysis revealed that eight PbGRF genes were significantly differentially expressed after treatment with different sugars; the expression of PbGRF7, PbGRF8, and PbGRF11 significantly increased, implying the involvement of these genes in sugar signaling. In addition, subcellular localization studies showed that the tested GRF proteins localize to the plasma membrane, and transgenic analysis showed that PbGRF18 can increase the sugar content in tomato leaves and fruit. The results of our research establish a foundation for functional determination of PbGRF proteins, and will help to promote a further understanding of the regulatory network in pear fruit development.

PheGRF4e initiated auxin signaling during moso bamboo shoot development.

BACKGROUND: As a ubiquitous acid-regulating protein family in eukaryotes, general regulatory factors (GRFs) are active in various life activities of plants. However, detailed investigations of the GRFs gene family in moso bamboo are scarce. METHODS AND RESULTS: Genome-wide characteristics of the GRF gene family in moso bamboo were analyzed using the moso bamboo genome. GRF phylogeny, gene structure, conserved domains, cis-element promoters, and gene expression were systematically analyzed. A total of 20 GRF gene family members were identified in the moso bamboo genome. These genes were divided into ε and non-ε groups. qRT-PCR (real-time quantitative reverse transcription polymerase chain reaction) showed that PheGRF genes responded to auxin and gibberellin treatment. To further study PheGRF gene functions, a yeast two-hybrid experiment was performed and verified by a bimolecular fluorescence complementation experiment. The results showed that PheGRF4e could interact with PheIAA30 (auxin/indole-3-acetic acid, an Aux/IAA family gene), and both were found to act mainly on the root tip meristem and vascular bundle cells of developing shoots by in situ hybridization assay. CONCLUSIONS: This study revealed that PheGRF genes were involved in hormone response during moso bamboo shoot development, and the possible regulatory functions of PheGRF genes were enriched by the fact that PheGRF4e initiated auxin signaling by binding to PheIAA30.

The relationship between bowling intensity and ground reaction force in cricket pace bowlers.

This study examined the relationship between perceived bowling intensity, ball release speed and ground reaction force (measured by peak force, impulse and loading rate) in male pace bowlers. Twenty participants each bowled 36 deliveries, split evenly across three perceived intensity zones: low = 70% of maximum perceived bowling effort, medium = 85%, and high = 100%. Peak force and loading rate were significantly different across the three perceived intensity zones in the horizontal and vertical directions (Cohen's d range = 0.14-0.45, p < 0.01). When ball release speed increased, peak force and loading rate also increased in the horizontal and vertical directions (ηp2 = 0.04-0.18, p < 0.01). Lastly, bowling at submaximal intensities (i.e., low - medium) was associated with larger decreases in peak horizontal force (7.9-12.3% decrease), impulse (15.8-21.4%) and loading rate (7.4-12.7%) compared to decreases in ball release speed (5.4-8.3%). This may have implications for bowling strategies implemented during training and matches, particularly for preserving energy and reducing injury risk.