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Article

The Relationship between Endogenous Hormone Content and Related Gene Expression and Tillering in Wild Kentucky Bluegrass

by
Xue Ha
,
Jinqing Zhang
,
Fenqi Chen
,
Yajun Li
and
Huiling Ma
*
Key Laboratory of Grassland Ecosystem, Ministry of Education, Pratacultural Engineering Laboratory of Gansu Province, Sino-U.S. Center for Grazingland Ecosystem Sustainability, College of Pratacultural Science, Gansu Agricultural University, Lanzhou 730070, China
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(12), 2899; https://doi.org/10.3390/agronomy13122899
Submission received: 2 November 2023 / Revised: 23 November 2023 / Accepted: 23 November 2023 / Published: 25 November 2023
(This article belongs to the Special Issue Advances in Genetics, Breeding, and Quality Traits in Forage and Turf Grass—2nd Edition)
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Abstract

:
Poa pratensis is widely distributed in cold temperate regions and can be used as a species for stress restoration and as a forage for livestock. Studying the genetic characteristics of tillering occurrence in bluegrass provides a theoretical basis for studying plant yield formation, environmental adaptation, and improving survival competitiveness. The regulating effects of endogenous hormone IAA content and the expression of related genes ARF1, ARF12, ARF14, ZT content and the expression of related genes CKX2, CKX3, CKX4, SL content and the expression of related genes D14-like, D14.1-like and D14 in wild Kentucky bluegrass were investigated. Kentucky bluegrass from Sunan and Qingshui was used to evaluate the influence of hormone and gene expression on tillering behavior. Endogenous hormone contents and expression levels of related genes in stems and roots of both materials were measured at prophase, peak, and anaphase of tillering. The results showed that among the three materials, the Sunan material had a better tillering ability for Poa pratensis, while the Qingshui material had poorer tillering ability. The downregulation of CKX2, CKX3, and CKX4 gene expression levels promotes the synthesis of ZT, thereby improving the tillering ability of the germplasm. Upregulation of ARF14, D14, and D14.1-like gene expression levels enhances the synthesis of IAA and SL, thereby inhibiting tillering. More importantly, the interaction between hormones affects the tillering ability of bluegrass, and high levels of ZT/IAA, ZT/SL, and ZT/(IAA+SL) values promote tillering. In summary, this study reveals the mechanism by which hormones regulate the occurrence of tillering in Kentucky bluegrass, providing a theoretical basis for understanding the genetic characteristics of plant type, effectively regulating tillering, studying yield development, environmental adaptation, and improving survival rate.

1. Introduction

Poa pratensis (Kentucky Bluegrass) is widely distributed in cold and temperate regions [1] and is an important component of prairie and meadow vegetation, as well as artificial grassland [2]. It has great potential for use in soil conservation, environmental restoration, and as a forage grass due to its strong regeneration ability, good turf quality, and high cold tolerance [3]. It is also an excellent forage for herbivorous livestock due to its palatability, nutritional value, and grazing tolerance. Tillering, which occurs at nodes and is independent of maternal growth, is an important morphological trait for plant survival and competition. It is a key breeding trait, and a major determinant of grass yield and overall production performance [4]. Tiller formation generally has two stages: axillary bud formation at the axils of each leaf, followed by axillary bud growth elongation [5]. Tiller development is important for the growth and development of Kentucky bluegrass, as well as for forage and seed yield. First, increasing the number of tillers in Kentucky bluegrass can effectively promote the utilization of soil nutrients by expanding the absorption range of plant roots and improving water and nutrient access [6], which in turn promotes the healthy growth of aboveground parts, thereby increasing the yield and nutritional value of forage [7]. In addition, increasing the tillering number helps plants quickly establish water and nutrient acquisition competitiveness, adapt to environmental stress [8], and increase forage grass stress resistance and adaptability [9]. Increasing the number of tillers can also expand the plant area, increase the coverage of the grassland, reduce the growth of weeds and surface exposure, and change the local ecological environment [10]. Tillering also increases the density and quality of the turf grass, thereby improving its trampling resistance and turf value [11]. The study showed that the number of tillers was significantly and positively correlated with the area of turfgrass cover and the above-ground biomass [12]. Understandably, the regulation of tiller production has been studied for several decades. However, current research on endogenous hormone regulation of tiller growth has mainly focused on crops such as rice, corn, and wheat, and research on the tiller growth characteristics of Kentucky bluegrass from the perspective of endogenous hormone regulation is limited. It has been shown that many factors, including developmental, genetic, endogenous hormonal, and environmental, influence tillering [13].
Major hormones include auxin (IAA), cytokinin (CK), and strigolactone (SL) [14], where IAA and SL inhibit tillering bud growth and CK promotes [15]. In transgenic rice plants, OsARF6 (AUXIN RESPONSE FACTOR 6), OsARF12, OsARF17, and OsARF25 genes were downregulated, resulting in a significant reduction in the number of tillers [16]. In contrast to IAA, CK regulates tillering and tillering development by controlling polar auxin transport in tillering buds [17]. In general, higher CK levels promote tillering in maize [18] and rice [19]. CK content is positively correlated with tillering bud elongation in wheat, rice, and barley [20]. CK, as an axillary bud growth promoter, can alleviate the inhibitory effect of IAA and break the dormant state of tillering buds. Overexpression of the CK degrading enzymes OsCKX4 (Cytokinin oxidase/dehydrogenase 4) gene can both reduce the number of tillers in rice [21]. SL, IAA, and CK together regulate tillering [22]. OsTB1 (OryzasativaTeosinteBranched 1) acts downstream of SL to inhibit rice tillering [23]. OsTB1 interacts with OsMADS57, inhibiting tillering by reducing transcription of SL receptor D14 (Differentiation 14) [24], the interaction of SL with D14 degrades D53, promoting the expression of OsTB1 [25]. The IAA transport channel hypothesis proposes that SL regulates tillering by regulating the amount and localization of the PIN1 (Personal Identification Number 1) protein on the plasma membrane [2]. The direct-action hypothesis refers to the regulation of SL by IAA, which acts as a second messenger of IAA and directly enters the axillary buds. Promoting the expression of SL synthesis genes positively regulates SL levels, inhibiting axillary bud growth [3].
Numerous studies have shown that the formation and development of tillers are mainly controlled by the levels of IAA, CK, and SL, and the balance between them [26]. Ferguson et al. [27] reported that SL inhibits the growth of lateral buds while CK promotes the growth of lateral buds. IAA regulates the growth of lateral buds by regulating the level of SL and low CK. Sibton et al. [28] demonstrated that the balance between IAA and CK plays a crucial role in axillary bud growth by genetically modifying the endogenous hormone balance in plants. In rice, IAA upregulates SL and downregulates CK biosynthesis through polar auxin transport, and SL moves vertically into the tiller buds and inhibits their growth [3], indicating that IAA inhibits lateral bud activation by reducing CK supply [29]. In addition, SL can reduce CK levels by inducing the expression of genes involved in CK catabolism, triggering a response from downstream genes, and thereby inhibiting rice tillering [30]. In this study, wild Kentucky bluegrass materials with different tillering characteristics were selected, and their tillering numbers were counted. The IAA content and expression levels of related genes ARF1, ARF12, ARF14, ZT content and their related genes CKX2, CKX3, CKX4, SL content and their related genes D14-like, D14.1-like, D14 expression levels were compared in the stems and roots of materials with different tillering abilities at the prophase, peak, and anaphase stages of tillering to reveal the mechanism by which hormones regulate the occurrence of tillering in Kentucky bluegrass. This, in turn, will allow us to understand how the genetic characteristics of plant type regulate the occurrence of tillering in Kentucky bluegrass, to design an appropriate tillering population, and provide a theoretical basis for research on plant yield formation, environmental adaptation, and improvement of survival competitiveness.

2. Materials and Methods

2.1. Plant Materials and Growth Condition

Wild Kentucky bluegrass germplasms were collected in Sunan, Qingshui, and Lanzhou, Gansu Province of China. The wild germplasm collected in Sunan is labeled as Sunan, those collected in Qingshui are labeled as Qingshui, and those collected in Lanzhou are labeled as Lanzhou (Supplementary Material: Table S1). We have permission to collect plant material. The voucher specimen, Sunan, PE 00977569, was identified by Yeqi He, and its sheet was deposited in the herbaria PE (http://sweetgum.nybg.org/ih/herbarium.php (accessed on 3 November 2023)), Lanzhou, N 019104175, was identified by Xianwu Kong and its sheet was deposited in the herbaria N (http://sweetgum.nybg.org/ih/herbarium.php (accessed on 3 November 2023)), Qingshui, WUK 0089157, was identified by Youwen Cui and its sheet was deposited in the herbaria WUK (http://sweetgum.nybg.org/ih/herbarium.php (accessed on 3 November 2023)), and they also could be searched on the Chinese Virtual Herbarium (https://www.cvh.ac.cn/spms/detail.php?id=f96f6165 (accessed on 3 November 2023)) and code (00977569, 019104175 and 0089157). Seeds of Kentucky bluegrass were germinated and cultured on moist filter paper at 25 °C for 10 days, and then the uniform seedlings were transferred into a hydroponic box (20 cm × 15 cm × 12 cm) filled with nutrient solution. Each germplasm was replicated three times and each box contained 6 seedlings. Plants were watered twice a week with half-strength Hoagland’s nutrient solution and grown in an environmentally controlled chamber with a day/night temperature of 25/20 °C, daily photoperiod cycle of 16 h (360 μmol·m−2·s−1), and relative humidity of 65%. At 56 d, 66 d, 76 d, 86 d, 96 d, and 106 d of transplanting, the number of tillers was observed and recorded (axial buds more than 1 cm are designed as tillers). The stems and roots were collected at 56 d (prophase), 76 d (peak), and 96 d (anaphase), immediately frozen in liquid nitrogen, and then stored in a refrigerator at −80 °C for RNA analysis and hormone extraction.

2.2. Measurement of IAA, ZT and SL Contents

For the extraction of endogenous hormones IAA, ZT, and SL, we weighed 1.0 g of frozen stems and roots at each stage of tillering (prophase, peak, and anaphase) before grinding to powder in liquid nitrogen. Approximately 0.2 g of ground sample was transferred to a 15 mL centrifuge tube, to which we added 6 mL of extraction solution (n-propanol:water:HCl = 2:1:0.002), tubes were to 4 °C (placed in an ice box), and agitated with a shaker for 30 min, all while protecting the sample from excess light. After removing the centrifuge tube, 3 mL of dichloromethane was added and the tubes were shaken again for 30 min. Samples were centrifuged at 4 °C 5000 r.min−1 for 5 min before 2 mL of supernatant (representing the organic phase layer solution) was removed into a new 15 mL centrifuge tube, which was then dried in the dark using liquid nitrogen. To the final product, we added 1 mL of 50% aqueous methanol solution to redissolve the freeze-dried supernatant before placing it on a 0.22 μm microporous membrane.
The supernatant was analyzed by high-performance liquid chromatography (HPLC-MS/MS). Plant hormones including ZT, IAA and SL, ZT and IAA were prepared in methanol for the external standard calibration curve (60 μg·mL−1, 30 μg·mL−1, 15 μg·mL−1, 7.5 μg·mL−1, 3.75 μg·mL−1, 1.88 μg·mL−1, 0.94 μg·mL−1, 0.47 μg·mL−1); SL hormones were prepared in methanol for the external standard calibration curve (1000 μg·L−1, 500 μg·L−1, 250 μg·L−1, 100 μg·L−1, 50 μg·L−1, 10 μg·L−1). The UPLC conditions were set as follows: Symmetry C18 chromatography column (4.6 mm × 250 mm, 5 μm) at a temperature of 30 °C. The mobile phase was A:B = methanol:0.1% phosphoric acid.

2.3. Determination of Transcription and Expression of Hormone Related Genes

Based on preliminary laboratory transcriptomic data (accession Number: PRJNA680673) [31], The Primer-BLAST of NCBI (http://www.ncbi.nlm.nih.Gov/tools/primer-blast (accessed on 3 November 2023)) was used to design the specific qRT-PCR primers of ARF1, ARF12, ARF14, CKX2, CKX3, CKX4, D14-like, D14.1-like and D14 genes, the primer sequences can be found in Table S1. Total RNA was extracted from 24 samples using the RNase Plant Mini kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the Prime Script RT Reagent Kit with a gDNA Eraser (perfect real-time) (TaKaRa, Osaka, Japan) according to the manufacturer’s instructions. qRT-PCR was performed on a StepOnePlus™ Real-Time PCR System (ABI) using SYBR premix Ex Taq (TaKaRa, Osaka, Japan). qRTPCR was performed on a StepOnePlus™ Real-Time PCR system (ABI) using SYBR premix Ex Taq (TaKaRa, Osaka, Japan).
Each 20 μL reaction system contained 2× SuperReal PreMix Plus 10 μL, ddH2O 3 μL, cDNA template 5 μL, and forward and reverse primer 2 μL. This was replicated three times for each sample. Using the Roche Light Cycler TM fluorescence quantitative PCR instrument (Roche, Rotkreuz, Switzerland), the PCR reaction conditions were as follows: Pre-denaturing was performed at 94 °C for 3 min; 40 cycles, denaturation was carried out at 94 °C for 10 s; annealing at 60 °C for 30 s; and extension at 72 °C for 30 s. Using the actin gene as an internal reference, the relative expression level of the gene was calculated using the 2−ΔΔCT formula [31].

2.4. Data Statistics

Results were analyzed using Excel 2013 and Spss 21.0 (IBM Corp., Armonk, NY, USA) [32] for Windows. We analyzed the data using an independent sample T-test and one-way ANOVA. We used Graph Pad Prism 8.0.2. software for drawing figures and conceptual diagrams. Data from each sampling event were analyzed separately.

3. Results

3.1. Study on Tillering Ability of Kentucky Bluegrass

There are significant differences in the axillary bud morphology of Kentucky bluegrass at different tillering stages. After 56 days of planting, Kentucky bluegrass began to tiller, and the trend of tillering per plant in each of the three bluegrass strains was basically consistent, all of which gradually increased with the growth of tiller buds Kentucky bluegrass had its most rapid increase in tillers from 76 to 96 days, with a relatively slow increase at the prophase of tillering followed by a rapid increase in tillers. Tillering rates peaked around 1.65 per 10 days, and at the anaphase, the growth of tillering number slowed down, fewer overall tillers appeared, wilting increased, and the population appeared to reach a stable number, no longer increasing. Among the different strains of Kentucky bluegrass, Sunan tended to have the highest tillering number, with an average of 2.97, followed by Lanzhou, while Qingshui had the lowest tillering number, with an average of 2.07 (Figure 1).

3.2. Comparison of Endogenous Hormone Levels in Stems and Roots of Sunan and Qingshui

The regulation of tillering of Poa pratensis by different hormones and gene expression is shown in Figure 2. To compare the effects of different endogenous hormone levels on the tillering ability of Kentucky bluegrass in Sunan and Qingshui materials, we measured the content of ZT, IAA, and SL in the stems and roots during the tillering, and found that there were significant differences in the contents of different types of endogenous hormones in materials with different tillering abilities (Figure 3). The ZT content in the stems of Sunan and Qingshui materials reached its peak at the prophase of tillering. During tillering, the ZT content of Qingshui material gradually decreased, reaching its lowest value at the anaphase of tillering. On the other hand, the ZT content of the Sunan materials reached its lowest value at the peak of tillering, and then it increased rapidly at the anaphase. In the roots, there is an inverse trend of variation in ZT content between the two materials, with ZT content gradually decreasing initially but reaching its peak at the anaphase of the tillering stage in Qingshui. Conversely, Sunan showed a gradual upward trend and reached its highest value at the anaphase of tillering. When comparing the two materials at the same period, compared to the prophase, the ZT content of Sunan materials is significantly higher than that of Qingshui at the peak and anaphase of tillering. In Sunan materials, the trend of the IAA content in the stems and roots is basically the same, first decreasing and then increasing, and reaching the minimum value at the peak of tillering. The main difference between Sunan and Qingshui is that the IAA content of Qingshui is significantly higher than Sunan at the peak and anaphase of tillering. In the stems, the variation trend of SL content in Sunan and Qingshui materials is basically the same. First decreasing and then increasing. SL levels of Qingshui are higher than that of Sunan. In the roots, the SL content of Qingshui decreases gradually, while the SL content of Sunan first decreases and then increases. In addition, the SL content of Qingshui material is significantly higher than that of Sunan at the prophase and peak of tillering. Increases in the level of ZT appear to be positively associated with the of tillers, while higher levels of IAA and SL are associated with a reduction in tillers.

3.3. Comparison and Ratio Analysis of Contents of Three Kinds of Endogenous Hormones

To further explore the relationship between the levels of three endogenous hormones and the occurrence of tillering in Kentucky bluegrass, we analyzed the levels and ratios of endogenous hormones at different tillering stages (Figure 4). It was found that during the tillering of Kentucky bluegrass, the ZT/IAA values of the two materials showed a similar trend in the stems, first decreasing and then increasing, but with different rates of change. In the roots, the ZT/IAA value of the Qingshui material shows a gradually decreasing trend. The Sunan material shows the opposite trend. In the Qingshui material, the ZT/SL value gradually decreases in the stems, and increases in the roots; whereas the ZT/SL values in the Sunan materials showed a trend of first increasing and then decreasing in both roots and stems, reaching their maximum value at the peak of tillering. The main difference is that the ZT/SL value in the Sunan is significantly higher than that in the Qingshui materials. At the roots, the ZT/(IAA+SL) value of Qingshui gradually decreases, while the Sunan gradually increases.

3.4. Correlation between Endogenous Hormone Levels and Tillering Strength

This study conducted a correlation analysis between hormone levels and tillering numbers at different tillering stages (Figure 5) and found that there was a highly significant positive correlation between IAA and SL content during the tillering stage. There is a highly significant positive correlation between the ZT content at the prophase of tillering and the ZT content at the peak and anaphase of tillering. The IAA and SL content at the anaphase of tillering and the SL content at the peak of tillering are significantly negatively correlated with tillers, and ZT content at the peak of tillering is significantly positively correlated with tillers. A correlation analysis was conducted between hormone content and tillering number in different issues (Figure S1). It was found that the IAA content in the stems was significantly negatively correlated with both ZT/IAA and ZT/(IAA+SL) content in the stems, as well as with the ZT, ZT/SL, ZT/(IAA+SL) content in the roots, and significantly positively correlated with the SL content in the roots. The IAA content in the roots is significantly positively correlated with the SL content in the stems and roots, and significantly negatively correlated with the ZT/IAA, ZT/SL, and ZT/(IAA+SL) content in the roots.

3.5. Expression Analysis of Hormone Synthesis-Related Genes in the Stems and Roots of Kentucky Bluegrass at Different Tillering Stages

3.5.1. Differential Expression of Genes Related to Auxin Signal Transduction

To further investigate the expression changes of genes related to plant tillering hormones in different tillering stages of Kentucky bluegrass, this study analyzed the differential expression of related genes based on the measurement of hormone IAA, CK, and SL content (Figure 6). At prophase, the expression trends of ARF1 and ARF12 genes in the two materials are basically the same. The stems and roots of the Qingshui material showed no significant changes at the peak and anaphase of tillering. In the stems of Sunan material, the ARF1 and ARF12 genes are almost not expressed at the peak of tillering, but they are significantly upregulated at the anaphase of tillering. In the roots, the two genes are downregulated at the peak and anaphase of tillering, and the degree of downregulation at the peak of tillering is greater than that at the anaphase of tillering. The expression of the ARF14 gene is inconsistent between the two materials. In the stems of the Qingshui material, the ARF14 gene showed almost no changes at the peak of tillering but was upregulated at the anaphase of tillering. In the roots, the ARF14 gene was downregulated at the peak and anaphase of tillering. In the stems and roots of Sunan, the ARF14 gene is downregulated at the peak of tillering, and downregulated in the stems at the anaphase of tillering, while upregulated in the roots. Compared to Qingshui, the ARF14 gene was upregulated in the stems of Sunan material at the prophase of tillering, and downregulated at tillering stages (Figure S2).

3.5.2. Differential Expression of CK Signal Transduction-Related Genes

The expression of CK-related genes CKX2, CKX3, and CKX4 in the two materials is shown in Figure 7. In the Qingshui material, three genes showed no significant changes during the tillering stage. In the stems of Sunan, CKX3 is upregulated at the peak and anaphase of tillering, with the difference being that the degree of upregulation at the anaphase is greater than that at the peak of tillering. Conversely, CKX3 is downregulated in the roots. CKX2 is upregulated in the stems of Sunan materials at the peak, not expressed at the anaphase of tillering, and downregulated at the peak and anaphase of tillering in the roots. In the stems of Sunan, the CKX4 gene is upregulated at the peak, and downregulated at the anaphase of tillering. In the roots, the CKX4 gene is downregulated at the peak and anaphase of tillering, and the degree of downregulation at the anaphase is greater than that at the peak. Compared to Qingshui, the CKX3 gene was upregulated in the stems of Sunan at the peak and anaphase of tillering, and in the roots, the gene was also upregulated at the prophase of tillering but downregulated at the peak and anaphase of tillering. In the stems of Sunan, the CKX2 gene is downregulated at the prophase and anaphase and upregulated at the peak of tillering. However, in the roots, CKX2 is upregulated at the prophase and downregulated at the peak and anaphase of tillering. In the stems of Sunan, the CKX4 gene is downregulated at the prophase and anaphase and upregulated at the peak of tillering. In the roots, it is upregulated at the prophase, downregulated at the peak and anaphase of tillering, and the degree of downregulation at the anaphase is greater than that at the peak of tillering (Figure S3).

3.5.3. Differential Expression of SL Signal Transduction-Related Genes

At prophase, the expression trends of SL-related genes D14-like, D14.1-like, and D14 in different stages of Qingshui and Sunan materials are basically consistent (Figure 8). Throughout tillering D14-like and D14.1-like were not expressed in the Qingshui material, but in the Sunan material, their expression was downregulated in the stems and roots at the peak and anaphase of tillering. Similarly, D14 gene expression was downregulated at the peak and anaphase of tillering in stems but upregulated at the peak and downregulated at the anaphase of tillering in roots. When comparing the two materials, the D14-like gene was upregulated in the stems of Sunan at the prophase and peak of tillering and downregulated at the anaphase of tillering. The D14.1-like gene is upregulated at the prophase, almost not expressed at the peak and anaphase of tillering. The D14 gene is upregulated. In the Sunan roots, the D14-like gene was upregulated at the prophase and downregulated at the peak and anaphase of tillering. Similarly, D14.1-like gene expression is upregulated in Sunan at the prophase and downregulated at the peak and anaphase of tillering. The D14 gene is almost not expressed at the prophase, upregulated at the peak of tillering, but downregulated at the anaphase of tillering (Figure S4).

4. Discussion

4.1. Tillering Characteristics and Research Significance of Wild Kentucky Bluegrass in Gansu Province

The seed yield of Kentucky bluegrass is made up of spikelets, number of seeds on spikelets, seed setting rate, and thousand kernel weight. Tiller is one of the most important agronomic traits that affects the number of spikelets. The quantity and quality of tillers directly affects the establishment of Kentucky bluegrass and is associated with the direction of development of other agronomic traits, ultimately affecting morphogenesis and yield [33]. One of the requirements for high Kentucky bluegrass yield is appropriate tillering. Too few tillers can be inadequate, while too many can cause adverse effects such as mass mortality, reduced seed set rate, and spikelet size, ultimately reducing yield. There have been systematic reports on the growth and developmental mechanisms of tillering in rice and wheat [34,35], but there are few research reports on the occurrence and regulation of tillering in Kentucky bluegrass. Thus, an understanding of the mechanism by which tillering occurs in Kentucky bluegrass can be used to improve yield and tolerance to climate and competition. As a major cool-season turfgrass species, Kentucky bluegrass varies in environmental adaptation and tillering potential [13]. Tillering in Kentucky bluegrasses is actually the result of lateral buds germinating at the base of the mother stem. Tillering ability is generally represented by tiller number, which refers to the number of near-ground branches in the plant. The tillers that can produce heads and fruits are called effective tillers, which determine the effectiveness of spikelets and the photosynthetic area of the plant and directly affect the yield [36]. The results of this study show that the tillering of Kentucky bluegrass occurs mostly at 56 days after seedling, and the tillering rate is the fastest from 76 to 96 days after the seedling germinates. Sunan had more tillers on average and a longer tillering window. The tillering ability of Qingshui is the weakest. The number of tillers increases initially and then remains stable. This indicates that the tillering ability of Sunan is stronger than that of Qingshui materials under the same planting conditions. Moreover, tillering characteristics such as starting time, tillering speed, and maximum tillers differ significantly among germplasms.

4.2. Relationship between Endogenous Hormone Content and Related Gene Expression in the Stems and Roots Kentucky Bluegrass and Tillering Formation at Different Developmental Stages

4.2.1. Relationship between IAA and Related Gene Expression and Tillering Formation and Development of Kentucky Bluegrass

IAA is the earliest hormone discovered to regulate plant tillering, synthesized in stem tips and tender leaves [37]. Its content, transportation, and distribution all affect the tillering of Kentucky bluegrass [38]. Mainly by generating apical dominance to inhibit the formation and development of tillering [39]. When the apical tissue of the plant is damaged or artificially removed, the apical advantage is weakened, and the dormancy of tillering buds is relieved, starting to germinate and grow. Studies have shown that auxin produced by the apical meristem and adjacent young leaves can inhibit the production of lateral buds [40]. The results of this study indicate that after the emergence of Kentucky bluegrass seedlings, the IAA content is the highest, and the growth point at the top of the stems grows upwards. With the occurrence of tillering, the IAA content gradually decreases, accompanied by the occurrence of large tillers in Kentucky bluegrass. This indicates that within a certain tillering time period, the tillers and IAA content of Kentucky bluegrass are significantly negatively correlated. The ARF family is an important part of the IAA response, distributed in various tissues and organs of plants, and can specifically combine with the auxin response element (AuxRE) “TGTCTC” therein to control and regulate the specific expression of IAA response genes, so as to achieve the control of plant growth and development [41]. Analysis of 20 representative TaARF genes in wheat showed that abnormal expression of TaARF11 and TaARF14 was the main reason for constraining dmc tillering [42]. Research on rice has found that overexpressing mir167 can decrease the expression levels of OsARF6, OsARF12, OsARF17, and OsARF25, and the number of tillers [16]. This experiment investigated the regulatory effects of the ARF gene and IAA content on the tillering of Poa pratensis. In the stems of Sunan, a material with strong tillering ability, IAA synthesis showed a trend of first decreasing and then increasing with the downregulation of the ARF14 gene expression level. In the roots, the expression level of ARF14 gene showed a trend of first decreasing and then increasing, while the synthesis of IAA showed the same trend. Mutants with OsARF11 and OsARF16 genes knocked out showed more tillers in rice [43]. In this study, when the expression of the ARF14 gene was down-regulated, the top-down Polar auxin transport of IAA was affected, resulting in the reduction of IAA accumulation in the stems and roots. A low concentration of IAA was conducive to the differentiation and development of protocells into axillary bud tissue and promoted the formation of new tiller buds [44]. However, in the stems of Qingshui associated with weak tillering ability, the amount of IAA synthesis first increases and then decreases as tillering proceeds. Tillering buds are inhibited as IAA synthesis increases in Kentucky bluegrass because high concentrations of IAA are not conducive to the formation of tillering buds. In the roots, when the expression level of the ARF14 gene is downregulated, the synthesis of IAA decreases. In the stems of Sunan materials, ARF1 and ARF12 genes are upregulated at the anaphase of tillering, while in the roots, ARF1 and ARF12 genes are downregulated at the peak and anaphase of tillering. It can be inferred that the ARF1 and ARF12 genes do not affect the formation and development of tillers during the process of tillering, and may be related to the occurrence of other agronomic traits in Kentucky bluegrass. In summary, IAA-related genes can affect the occurrence of plant tillering by regulating hormone levels. ARF14, as a response factor in the IAA signaling pathway, may be located upstream of the IAA metabolic pathway. By sensing the IAA signal in the environment, it can affect the production of IAA transporters, regulate the Polar auxin transport of IAA, reduce/promote the accumulation of IAA in the stems and roots, and thus promote/inhibit the differentiation of protocells into axillary bud tissue [45].

4.2.2. Relationship between CK and Related Gene Expression and Tillering Formation and Development of Kentucky Bluegrass

CK is synthesized in the roots of the plant and then transported from the roots to the stems, directly promoting the activation of axillary buds [46]. Numerous studies have shown that CK plays an important regulatory role in the growth and development of lateral buds [47]. Yuan et al. found that directly applying CK to the lateral buds of the decapitated Vicia app mutant increased the production of lateral buds [48]. In the study of wheat, it was found that the occurrence and disappearance of tillers are closely related to the content of ZT+ZR in tillering nodes [49]. ZT plays an important role in the tillering process. CK, as a plant hormone promoting the growth of axillary buds, can not only relieve the inhibition of IAA on tillering buds but also break the dormancy of tillering buds. Cytokinin oxidation/dehydrogenase (CKX) is the only known oxidase that can degrade CK, and it is the main factor controlling the degree of plant cell division [50]. This study found that the transcriptional expression levels of CKX2, CKX3, and CKX4 genes in Sunan were higher than those in Qingshui. Research on rice has found that specifically reducing the expression levels of OsCKX2 and CKX4 significantly increases the number of tillers [51]. It can be seen that ZT mainly regulates the expression of tillering genes in plant tillering buds to achieve rapid growth of tillering buds, which also provides evidence for the results of this experiment. Research on wheat has shown that the expression of CKX3 and CKX5 is negatively correlated with the number of tillers [52]. This study found that as tillering occurs in Sunan, the expression levels of CKX2, CKX3, and CKX4 genes increase in the stems, while decreasing in the roots. It indicates that the transcriptional expression levels of CKX2, CKX3, and CKX4 genes are closely related to tillering formation. ZT content decreases with the increase in CKX2, CKX3, and CKX4 gene transcriptional expression levels, which hinders the formation of tillering buds, but the initiation of tillering buds is not affected, which is similar to previous research results [53]. We can speculate that ZT regulates the germination and the dormancy of tillering buds comes from the roots, not the stems, in Kentucky bluegrass. At the prophase of tillering, the roots synthesize a large amount of ZT for rhizome expansion, and the content of ZT transported to the stems is less. With the occurrence of tillering, the synthetic ZT gradually increases, and the root’s activity is vigorous. Redundant ZT is transported to stems through Polar auxin transport to promote a large number of tillers.

4.2.3. Relationship between SL and Related Gene Expression and Tillering Formation and Development of Kentucky Bluegrass

SL is mainly synthesized at the roots, then inhibiting the growth of tillering buds [54]. Currently, SL has been isolated from many plants. Gomez Rhodan et al. and Umehara et al. simultaneously and independently revealed the relationship between the content of SL compounds and increased branching in multi-branched mutants of Pea, Rice, and Arabidopsis. A decrease in SL content leads to an increase in branching, and applying SL in vitro can salvage the mutant phenotype of plants [55,56]. Xu et al. conducted an experiment using conventional rice varieties’ Yangdao 6 ‘and’ Nanjing 44 ‘as materials where they applied the synthetic compound GR24 of SL to the roots during the tillering stage, the results showed that applying trace amounts of (2 μmol·L) can have a good inhibitory effect on rice tillering, with a decrease in tillering incidence of more than 95 percentage points compared to the control. These experiments clearly indicate that SL inhibits plant branching [57]. In this study, Sunan, a material with strong tillering ability, had significantly lower SL content than Qingshui. This may be one of the reasons why Sunan has stronger tillering ability than the Qingshui material. Research on rice has found that the D14 gene encodes an α/β folding hydrolase, and the D14 protein may be a receptor for SL [58]. The dwarf multi-tillering rice mutant DHT1 mutation results in blocked transcription and splicing of D14 precursor mRNA, reduced D14 protein, hindered SL signal transmission, and ultimately leads to the accumulation of D53 protein, an inhibitor of SL signaling pathway, promoting tillering [59]. In this study, the D14 gene was downregulated in the stems of Sunan with strong tillering ability at the peak and anaphase of tillering, but upregulated in the roots at the peak and downregulated at the anaphase of tillering. We speculate that the D14 gene in the stems is closely related to the formation and development of tillering, while D14 in the roots may be related to the development of root hairs and the ability of rhizomes to expand. In rice, the OsD3 gene can be assembled into a SCFD3 complex that, in combination with OsD14, inhibits rice tillering [60]. OsMADS57 inhibits the expression of the OsD14 gene by combining the cArG motif of the promoter, thus increasing rice tillering [61], indicating that MADS can affect the tillering of gramineae through SL. Our research team identified the PpMADS gene [62] during inflorescence development through transcriptome in the early stage, and can further study the regulation mechanism of SL on tillering of Kentucky bluegrass by starting with MADS gene in the later stage. The above-mentioned tillering-related genes play different roles in the process of tillering formation, including genes that directly affect the formation of tillering buds and indirectly regulate tillering buds through genes that affect hormone synthesis. In this experiment, the expression levels of D14-like and D14.1-like genes were closely related to tillering formation, and an increase in gene expression levels promoted SL synthesis, thereby inhibiting tillering formation, this is similar to previous research results. The functional loss of TN1 and TIF1 can cause downregulation of the key gene D14 in rice tillering regulation. Moreover, excessive expression of D14 in the TN1-1 mutant can significantly reduce tillering, further indicating that D14 affects rice tillering formation by affecting SL synthesis [24]. In summary, there is a specific relationship between the formation and development of tillers and the content of hormones in stems and roots in Kentucky bluegrass. However, due to the different environmental factors affecting the stems and roots, the changes in hormone levels in the roots are not as significant as in the stems, but can also reflect the impact of different endogenous hormones on tillering.

4.3. Effects of Dynamic Equilibrium of Endogenous Hormones of Kentucky Bluegrass on Tillering

The tillering process of Kentucky bluegrass is a complex process where IAA, CK, and SL interact to form a complex regulatory network [63]. The causes of tillering formation are more often caused by changes in hormone balance [13]. Brenner et al. proposed that the essential role played by assimilating transport allocation in tiller development is mediated by balancing endogenous hormones [64]. Therefore, when controlling tillering by regulating endogenous hormones, one should not only focus on changing the content of a certain hormone, but also adjust the balance relationship of endogenous hormones in multiple ways to reduce the occurrence of ineffective tillering, improve the quality and quantity of tillering and spikelet formation, and achieve the goal of increasing yield. Research has shown that IAA indirectly inhibits plant tillering through the second messenger, and CK and SL are important components of the second messenger. IAA not only inhibits the synthesis of CK, but also promotes the synthesis of SL, and CK can also inhibit local synthesis of SL [65]. Morris proposed that IAA indirectly inhibits the growth of lateral buds by inhibiting the formation of CK [66]. This study found that the tillers were significantly positively correlated with ZT/IAA, ZT/SL, and ZT/(IAA+SL) value in the roots, extremely significantly negatively correlated with IAA content, and significantly negatively correlated with the SL content in the stems. This indicates that IAA and SL play a dominant role in the inhibition of tillering buds during the process of tillering in Kentucky bluegrass, and suggests there is a positive correlation between the two, but CK promotes the growth of tillering buds. The relative content of IAA and CK is a key factor in regulating the growth of lateral buds, and increasing the CK/IAA value can promote the germination of tillering buds. It is speculated that the IAA regulates tillering growth by regulating CK content. Upregulation of CKX expression generally leads to a decrease in CK content, and IAA induces CK degradation by regulating the expression of CKX genes [67]. OsCKX9 can be induced by SL to express and degrade CK in plants, increase the tillering number, reduce plant height, and affect spike size, thereby improving rice productivity [68]. On the other hand, IAA also inhibits the formation of rice tillers by promoting SL synthesis [69]. This study suggests that the ratio of IAA and CK, rather than their absolute concentration, regulates lateral growth. Research on corn has shown that high CK/IAA and CK/ABA values promote tillering, while low CK/IAA and CK/ABA values inhibit tillering [70]. The results of this study indicate that in materials with strong tillering ability, the ZT/IAA value shows a trend of first decreasing and then increasing, while the ZT/SL value shows a trend of first increasing and then decreasing. As tillering occurs, the ZT/(IAA+SL) value in the stems gradually increases, which is consistent with the pattern of weak to strong and then weak tillering ability. Moreover, the stronger the tillering ability, the higher the ZT/IAA, ZT/SL, and ZT/(IAA+SL) values. Therefore, high ZT/IAA, ZT/SL, and ZT/(IAA+SL) values are beneficial for tillering occurrence, while conversely, they inhibit it.

4.4. Strategies of Applying Plant Hormone to Improve Tillering of Kentucky Bluegrass

In Helianthus annuus and Zea mays, reducing the branching (tillering) number may be necessary to improve the aboveground structure and improve seed yield efficiency [71]. In grass plants such as Eremochloa opiuroides [72] and Medicago truncatula [73], an increased number of tillers or branches is associated with increased seed yield, grass yield, and bare land coverage. The application of exogenous hormones can effectively reduce or increase tillering, meeting practical needs. In recent years, the regulatory mechanisms of various hormones on tillering have been clarified. For example, exogenous application of IAA, SL, GA, ABA, and BR can reduce branching/tillering, CK inhibitor treatment can also reduce branching/tillering, but exogenous CK treatment can increase plant branching. External application of CK, increases the content of endogenous hormone 6-BA in tillering nodes, thereby promoting the growth and development of tillering buds in rice, suggesting that high CK content promotes tillering [74]. Harrison et al. [75] found that applying IAA externally to oats inhibited the growth and development of their tillering buds. In this study, we found that endogenous hormones and the expression of their related genes can regulate the tillers of Kentucky bluegrass. The decrease in ZT and the increase in IAA and SL content are the reasons for inhibiting the growth and development of axillary buds and high yield. Therefore, the focus of future research is to explore the regulatory mechanism of exogenous hormones, as well as the optimal application concentration, application frequency, application time, and application location of related hormones, with the aim of providing a basis for targeted regulation of tillering occurrence in Kentucky bluegrass.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13122899/s1, Table S1. Sequence of gene primers used in qRT-PCR. Table S2. Sequence of gene primers used in qRT-PCR. Figure S1. Correlation between ZT, IAA, SL, ZT/IAA, ZT/SL, ZT/(IAA+SL) content and per plant tiller number in stems and roots during bluegrass tiller growth period. Figure S2. Analysis of the expression patterns of ARF1, ARF14, and ARF12 genes in the stems (A,C,E) and roots (B,D,F) of Kentucky bluegrass at the same period. Figure S3. Analysis of the expression patterns of CKX3, CKX2, and CKX4 genes in the stems (A,C,E) and roots (B,D,F) of Kentucky bluegrass at the same period. Figure S4. Analysis of the expression patterns of D14-like, D14.1-like, and D14genes in the stems (A,C,E) and roots (B,D,F) of Kentucky bluegrass at the same period.

Author Contributions

H.M. conceived the original research plans, supervised the experiments, provided funding support, and agreed to serve as the author responsible for contact and communication. X.H., J.Z. and Y.L. performed the experiments. X.H., J.Z. and F.C. analyzed the data and wrote the article. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (project #31760699).

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, and further inquiries can be directed to the corresponding authors.

Acknowledgments

We would like to thank Daniel Petticord at the University of Cornell for his assistance with the English language and grammatical editing of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Dynamic changes in tillering number and morphology of axillary buds in Kentucky bluegrass germplasm. (A) shows the changes in the number of tillers from the beginning to the end of tillering in Lanzhou, Qingshui, and Sunan germplasms. The result is the average tillering number of 12 plants, calculated every 10 days. (B) shows the morphological characteristics of seedling tillering buds. The figure shows the morphological characteristics of tillering buds of seedlings in the prophase tillering stage of Sunan germplasm. (Canon, EOS200D, DaLian, China), and the red arrow indicates the tillering buds.
Figure 1. Dynamic changes in tillering number and morphology of axillary buds in Kentucky bluegrass germplasm. (A) shows the changes in the number of tillers from the beginning to the end of tillering in Lanzhou, Qingshui, and Sunan germplasms. The result is the average tillering number of 12 plants, calculated every 10 days. (B) shows the morphological characteristics of seedling tillering buds. The figure shows the morphological characteristics of tillering buds of seedlings in the prophase tillering stage of Sunan germplasm. (Canon, EOS200D, DaLian, China), and the red arrow indicates the tillering buds.
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Figure 2. Plant hormones and their related genes regulate tillering of Poa pratensis in grassland, arrows indicate positive regulation, lines indicate negative regulation. The solid line represents the results obtained from this study, while the dashed line represents the results inferred from this study. Red arrows indicate tillering buds.
Figure 2. Plant hormones and their related genes regulate tillering of Poa pratensis in grassland, arrows indicate positive regulation, lines indicate negative regulation. The solid line represents the results obtained from this study, while the dashed line represents the results inferred from this study. Red arrows indicate tillering buds.
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Figure 3. Changes in endogenous ZT, IAA, and SL during Kentucky bluegrass tillering growth period. (A,C,E): endogenous ZT, IAA, and SL in stems, (B,D,F): endogenous ZT, IAA, and SL in roots. Values are the mean ± SD. Different letters at each point (a, b, c, etc.) indicate a pairwise comparison in hormones of different germplasm at the same tillering stage by least significant difference test (p ≤ 0.05). Asterisks indicate extremely significant. “*”Indicates significant, “**” indicates extremely significant.
Figure 3. Changes in endogenous ZT, IAA, and SL during Kentucky bluegrass tillering growth period. (A,C,E): endogenous ZT, IAA, and SL in stems, (B,D,F): endogenous ZT, IAA, and SL in roots. Values are the mean ± SD. Different letters at each point (a, b, c, etc.) indicate a pairwise comparison in hormones of different germplasm at the same tillering stage by least significant difference test (p ≤ 0.05). Asterisks indicate extremely significant. “*”Indicates significant, “**” indicates extremely significant.
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Figure 4. Changes in endogenous hormones ZT content and mass ratios of various hormones during Kentucky bluegrass tillering growth period. (A,C,E): endogenous ZT content and mass ratios of various hormones in stems, (B,D,F): endogenous ZT content and mass ratios of various hormones in roots. Different letters at each point (a, b, c, etc.) indicate a significant pairwise comparison by least significant difference test (p ≤ 0.05). Asterisks indicate extremely significant. “*”Indicates significant, “**” indicates extremely significant.
Figure 4. Changes in endogenous hormones ZT content and mass ratios of various hormones during Kentucky bluegrass tillering growth period. (A,C,E): endogenous ZT content and mass ratios of various hormones in stems, (B,D,F): endogenous ZT content and mass ratios of various hormones in roots. Different letters at each point (a, b, c, etc.) indicate a significant pairwise comparison by least significant difference test (p ≤ 0.05). Asterisks indicate extremely significant. “*”Indicates significant, “**” indicates extremely significant.
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Figure 5. Correlation between the number of tillers and contents of IAA, SL, and ZT of Kentucky bluegrass at different tillering stages. Red indicates positive correlation, blue indicates negative correlation, and the darker the color, the stronger the correlation “*”and “**” indicate significant correlations at 0.05 and 0.01 confidence levels (two-tailed), respectively.
Figure 5. Correlation between the number of tillers and contents of IAA, SL, and ZT of Kentucky bluegrass at different tillering stages. Red indicates positive correlation, blue indicates negative correlation, and the darker the color, the stronger the correlation “*”and “**” indicate significant correlations at 0.05 and 0.01 confidence levels (two-tailed), respectively.
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Figure 6. The expression patterns of ARF1, ARF14, and ARF12 in stems (A,C,E) and roots (B,D,F) of Kentucky bluegrass. Different letters a, b, and c indicate significant differences in the same material at different periods.
Figure 6. The expression patterns of ARF1, ARF14, and ARF12 in stems (A,C,E) and roots (B,D,F) of Kentucky bluegrass. Different letters a, b, and c indicate significant differences in the same material at different periods.
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Figure 7. Expression patterns of CKX3, CKX2, and CKX4 genes in stems (A,C,E) and roots (B,D,F) of Kentucky bluegrass. Different letters a, b, and c indicate significant differences in the same material at different periods.
Figure 7. Expression patterns of CKX3, CKX2, and CKX4 genes in stems (A,C,E) and roots (B,D,F) of Kentucky bluegrass. Different letters a, b, and c indicate significant differences in the same material at different periods.
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Figure 8. The expression patterns of D14 like, D14.1 like, and D14 gene in stems (A,C,E) and roots (B,D,F) of Kentucky bluegrass. Different letters a, b, and c indicate significant differences in the same material at different periods.
Figure 8. The expression patterns of D14 like, D14.1 like, and D14 gene in stems (A,C,E) and roots (B,D,F) of Kentucky bluegrass. Different letters a, b, and c indicate significant differences in the same material at different periods.
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Ha, X.; Zhang, J.; Chen, F.; Li, Y.; Ma, H. The Relationship between Endogenous Hormone Content and Related Gene Expression and Tillering in Wild Kentucky Bluegrass. Agronomy 2023, 13, 2899. https://doi.org/10.3390/agronomy13122899

AMA Style

Ha X, Zhang J, Chen F, Li Y, Ma H. The Relationship between Endogenous Hormone Content and Related Gene Expression and Tillering in Wild Kentucky Bluegrass. Agronomy. 2023; 13(12):2899. https://doi.org/10.3390/agronomy13122899

Chicago/Turabian Style

Ha, Xue, Jinqing Zhang, Fenqi Chen, Yajun Li, and Huiling Ma. 2023. "The Relationship between Endogenous Hormone Content and Related Gene Expression and Tillering in Wild Kentucky Bluegrass" Agronomy 13, no. 12: 2899. https://doi.org/10.3390/agronomy13122899

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