Abstract
Microbial fertilizers can activate and promote nutrient absorption and help inflorescence elongation. To understand the molecular mechanisms governing grape (Vitis vinifera) inflorescence elongation after microbial fertilizer application, we comprehensively analyzed the transcriptome dynamics of ‘Summer Black’ grape inflorescence at different leaf stages. With the development of ‘Summer Black’ grape inflorescence, gibberellic acid content gradually increased and was clearly higher in the microbial fertilizer group than in the corresponding control group. In addition, the microbial fertilizer and control groups had 291, 487, 490, 287, and 323 differentially expressed genes (DEGs) at the 4-, 6-, 8-, 10-, and 12-leaf stages, respectively. Kyoto Encyclopedia of Genes and Genomes pathway annotation revealed that most upregulated DEGs were enriched in starch and sucrose metabolism pathways at the 6-, 8-, and 10-leaf stages. Weighted gene coexpression network analysis identified stage-specific expression of most DEGs. In addition, multiple transcription factors and phytohormone signaling-related genes were found at different leaf stages, including basic helix-loop-helix proteins, CCCH zinc finger proteins, gibberellin receptor GID1A, 2-glycosyl hydrolases family 16, protein TIFY, MYB transcription factors, WRKY transcription factors, and ethylene response factor, suggesting that many transcription factors play important roles in inflorescence elongation at different developmental stages. These results provide valuable insights into the dynamic transcriptomic changes of inflorescence elongation at different leaf stages.
The inflorescence architecture, especially inflorescence length, significantly affects crops’ yield and stability (Jiang et al. 2011). Studies have shown that increasing inflorescence length is essential for improving the yields of soybean [Glycine max (Benlloch et al. 2015)], arabidopsis [Arabidopsis thaliana (Bradley et al. 1997)], petunia [Petunia hybrida (Souer et al. 1998)], rugosa rose [Rosa rugosa (Kawamura et al. 2011)], and maize [Zea mays (Bomblies et al. 2003)]. Gibberellin is well known for its growth control function in flower, fruit, and seed development. Therefore, exogenous gibberellic acid (GA) application plays an important role in viticulture (Cheng et al. 2015). Moreover, exogenous gibberellin is often applied in the first week of flowering to reduce flower ear density and promote flower inflorescence elongation in grape (Vitis vinifera) production and cultivation. Further, exogenous gibberellin treatment enhances grape inflorescence elongation, improves fruit ear appearance, reduces fruit diseases, and promotes fruit quality. However, our investigation of grape cultivation in the Guangxi area of China found that improper use of gibberellin could lead to grape distortion, fruit stiffness, and stem hardening. More seriously, it also causes overgrowth of new shoots and inhibition of winter bud development and differentiation, thus affecting grape flowering in the next season. Therefore, developing alternative fertilizers is of great interest to avoid the severe disadvantages of improper use of plant growth regulators in grape planting.
Microbial fertilizers are fermented products of single microbial inoculants of various strains with specific functions (Cheng et al. 2015). They are rich in nutrients and physiologically active substances and could promote plants’ nutrient absorption and resistance to pests and diseases (Ku et al. 2018). So far, microbial fertilizers have played important roles in the sustainable development of Chinese agriculture. Therefore, developing microbial fertilizers has attracted more attention worldwide and has become a research hotspot in biotechnology and agricultural production development (Liu G et al. 2019). We have applied microbial fertilizers in vineyard production and found that proper application of microbial fertilizers significantly promoted inflorescence elongation to its ideal length without hormone treatment (Chen et al. 2021), thus avoiding the side effects of hormone treatment such as fruit sclerosis and effectively improving grape quality. However, the molecular mechanisms under these advances remain poorly defined. Therefore, we performed inflorescence transcriptomic analysis after applying microbial fertilizers to reveal the mechanisms underlying microbial fertilizers’ promoting effects on grape inflorescence elongation and provide technical and theoretical support for grape cost-saving and efficiency-based cultivation.
Materials and Methods
Plant treatments.
The experiment was carried out in the double-season grape demonstration garden of the Academy of Agricultural Sciences of Guangxi Zhuang Autonomous Region (Nanning, Guangxi, China). The grape cultivar tested was 3-year-old ‘Summer Black’ grape, and the fertilizers tested were the seaweed-derived polypeptide fertilizer and 120-preservative microbial agents from Beijing Zhongnong Fuyuan Bioengineering Technology Co., Ltd. (Tongzhou, Jiangsu, China), with a nitrogen content of 12.59% and 0.59%, respectively. The fermented sheep manure from Xi'an Heidi Fertilizer Co., Ltd. (Xi'an, Shaanxi, China) has a nitrogen content of 1.16%. According to the principle of equal nitrogen amount, seaweed-derived polypeptide fertilizer and 120-preservative microbial agents at 1125 kg·ha−1 each and 5250 kg·ha−1 sheep manure were applied to the experimental plot (T) and sheep manure at 17,954.85 kg·ha−1 was applied to the control plot (CK). Before pruning in the spring, the fertilizer mixture was applied to the fertilizer ditch as the base fertilizer and covered compactly with the soil. Each treatment had three replicates with 10 grape plants per replicate. Five to six inflorescences were collected at 4-, 6-, 8-, 10-, and 12-leaf stages in each plot with three biological replicates, representing T1, T2, T3, T4, and T5 groups at the experimental plot and CK1, CK2, CK3, CK4, and CK5 groups in the CK plot. Meanwhile, corresponding leaf samples in the T1, T2, T3, CK1, CK2, and CK3 groups were collected and snap-frozen in liquid nitrogen. The length of grape inflorescence at the full flowering stage was measured.
Determination of gibberellic acid (GA3) content.
GA3 content in the young leaves and inflorescences of T1, T2, T3, CK1, CK2, and CK3 groups were determined using high-performance liquid chromatography [HPLC (Rigol L3000; Beijing Puyuan Jingyi Technology Co., Ltd., Changping, Beijing, China)]. Briefly, 1-g samples were ground in a mortar and soaked in 1 mL of precooled 80% methanol overnight at 4 °C. After centrifugation at 8000 gn for 10 min, the residues were soaked in 0.5 mL reagent for another 2 h. After centrifugation, the supernatants were taken, combined, and blow-dried at 40 °C using nitrogen to remove the organic phase. The sample was then extracted and decolored using 0.5 mL reagent three times. After discarding the upper ether phase, the lower aqueous phase was collected and adjusted to pH 2.8. The same process was performed four times for each sample. An appropriate amount of solution was filtered in the sample bottle with a pinhead filter and used to determine GA3 content by HPLC. In brief, 10-μL samples were injected into a C18 reversed-phase chromatographic column [250 mm × 4.6 mm, 5 μm (Kromasil; Beijing Huideyi Technology Co., Ltd., Changping, Beijing, China)] and separated at 30 °C by using a mixture of 35 parts of mobile phase A (formaldehyde) and 65 parts of B (0.1% phosphoric acid aqueous solution at the flow rate of 1 mL·min−1. Samples were detected at 210 nm for 40 min. GA3 content in plant samples (micrograms per gram) was calculated as the detection concentration (micrograms per milliliter) × dilution volume (milliliters)/weighing mass (grams), where the dilution volume was the volume of solution used when the sample was finally dissolved, and the weighing mass was the sampling mass at the time of extraction. The detection concentration was calculated automatically by the instrument based on the peak area of the substance to be measured.
Measurement of grape inflorescence length.
At the full flowering stage, the inflorescence was measured with a ruler. The petiole length was defined as the length from the place where the inflorescence was born to the accessory spike. The flower spike length was defined as the length from the accessory spike to the inflorescence spike tip. Total inflorescence length was defined as the length from where inflorescence was born to the spike tip. Thirty inflorescences were randomly measured for each repetition, and the average value was calculated.
RNA isolation, complementary DNA library preparation, transcriptome sequencing, RNA sequencing and data analysis, and quantitative real-time polymerase chain reaction analysis.
Total RNAs were extracted from each inflorescence sample using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) library preparation and transcriptome sequencing were conducted as previously reported (Liu H et al. 2015; Zhong et al. 2011). Clean reads were mapped to the grape reference genome using TopHat2 software (Goldstein et al. 2016), and only unique mapping reads were used to calculate gene expression. RNA-seq data were analyzed as previously described (Babarinde et al. 2019). Differentially expressed genes (DEGs) were identified using the edge package (Robinson et al. 2010) with a false discovery rate (FDR) <0.05 and an absolute log2 ratio ≥1. To validate the accuracy of RNA sequencing (RNA-seq), quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed using SYBR Premix Ex Taq (Tli RNase H Plus) master mix (Takara-Bio, Shiga, Japan) following the manufacturer’s instructions (Applied Biosystems 7500 Fast Real-Time PCR System; Thermo Fisher Scientific, Waltham, MA, USA). The PCR amplification program was 15 s at 95 °C followed by 40 cycles of 30 s at 95 °C and 60 s at 60 °C. Fold changes in gene expression level were calculated using the 2-ΔΔCt method (Li et al. 2020). The heatmap of DEGs was clustered using the BMK cloud (Beijing Baimaike Cloud Technology Co., Ltd. Shunyi District, Beijing, China). Gene Ontology [GO (Gene Ontology Consortium 2000)] enrichment analyses of the DEGs were performed to understand the biological significance of the DEGs using the GOseq R package. GO terms were considered significantly enriched with a corrected P < 0.05 (Young et al. 2010).
Statistical analysis.
All the data were analyzed using statistical software (IBM SPSS Statistics ver. 20.0; IBM Corp., Armonk, NY, USA). The normality of distribution and homogeneity of variances were examined using Shapiro–Wilk’s and Levene’s tests, respectively. Differences among groups were compared using the one-way analysis of variance. P < 0.05 was considered statistically significant.
Results
Changes in inflorescence length among different groups.
As shown in Table 1, the difference in spike stalk length between the two treatments was not obvious, but the difference in spike length and total inflorescence length between the microbial fertilizer group and CK group was significant, indicating that the application of microbial fertilizers effectively elongated the inflorescence of ‘Summer Black’ grape.
The inflorescence length of ‘Summer Black’ grape at full flowering stage under fertilizer treatment (T) and control treatment (CK).
Changes in GA3 content among different groups.
As shown in Fig. 1, GA3 contents in leaves and inflorescence increased gradually with ‘Summer Black’ grape development and were evidently higher in the microbial fertilizer group than in the corresponding CK group, indicating that the application of microbial fertilizers effectively improved gibberellin content in leaves and inflorescence (Fig. 1). Consequently, the increased gibberellin could promote grape inflorescence elongation, improve the appearance quality of fruit ear, reduce the incidence of fruit diseases, and improve fruit quality (Wang et al. 2019; Yang et al. 2020).
Gibberelli acid (GA3) content of ‘Summer Black’ grape in inflorescences (A) and leaves (B) under fertilizer treatment and control treatment. *P < 0.05.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Transcriptomic analysis of grape inflorescence at different leaf stages.
Global transcriptomic changes in ‘Summer Black’ grape inflorescence at the 4-, 6-, 8-, 10-, and 12-leaf stages were examined. A total of 202.7 Gb of clean data were screened from 30 inflorescence samples, each containing ≥5.8 Gb of data with quality value scores of 30 (Q30) more than 93.19% (Supplemental Table 1). Subsequently, clean reads of each sample were aligned to the reference genome (Canaguier et al. 2017), with the comparison efficiency ≥81.53% (Supplemental Table 2). DEGs screening based on FDR <0.05 and absolute log2 ratio ≥1 revealed 291, 487, 490, 287, and 323 DEGs, including 125, 322, 235, 146, and 138 upregulated genes and 166, 165, 255, 141, and 185 downregulated genes, between the experimental and corresponding CK groups at 4-, 6-, 8-, 10-, and 12-leaf stages, respectively (Fig. 2). Moreover, DEGs at 4-, 6-, 8-, 10-, and 12-leaf stages in the microbial fertilizer group were clustered away from those in the corresponding CK group, indicating that the application of microbial fertilizers altered gene expression.
The number of differentially expressed genes (DEGs) in inflorescences of ‘Summer Black’ grape in the comparisons between microbial fertilizer-treated and nontreated groups. The DEGs were identified using a false discovery rate <0.05 and an absolute log2 ratio ≥1.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Kyoto Encyclopedia of Genes and Genomes pathway analysis of DEGS.
All DEGs at different leaf stages were subjected to Kyoto Encyclopedia of Genes and Genomes pathway analysis to explore the involved major metabolic pathways. As shown in Supplemental Fig. 1, the upregulated DEGs at the four-leaf stage were mainly enriched in sesquiterpenoid and triterpenoid biosynthesis, amino sugar and nucleotide sugar metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, and starch and sucrose metabolism (Supplemental Fig. 1). The downregulated DEGs at the four-leaf stage were enriched in phenylpropanoid biosynthesis, starch and sucrose metabolism, and plant hormone signal transduction (Supplemental Fig. 2). The majority of upregulated DEGs at the 6-, 8-, and 10-leaf stages were enriched in starch and sucrose metabolism (Supplemental Figs. 3–5). The majority of the upregulated DEGs at the 12-leaf stage were enriched in the phenylpropanoid biosynthesis pathway and starch and sucrose metabolism (Supplemental Fig. 6).
Identification of conserved and/or divergent gene co-expression modules.
To investigate the gene regulatory network (GRN) at different stages, we identified coexpressed gene sets using weighted gene coexpression network analysis. A total of 10 coexpression modules or major subnetworks representing interactions among genes with similar expression profiles were identified (Fig. 3A). Further, we associated each coexpression module with corresponding developmental stages using Pearson correlation coefficient analysis (Fig. 3B) and found many modules were correlated with more than one development stage, although some were only correlated with a specific leaf stage. For example, the blue module was specifically correlated with the T1 stage (r ≥ 0.8), and the green and midnight blue modules were specifically correlated with the T5 stages (Fig. 3).
Coexpression networks at different developmental stages of ‘Summer Black’ grape. (A) Hierarchical clustering tree (dendrogram) of differentially expressed genes based on coexpression network analysis. (B) Module-tissue association. Each row and column correspond to a module and a specific tissue, respectively. The color of each cell at the row–column intersection indicates the correlation coefficient between the module and the tissue type. Dark red or dark green indicates a high degree of correlation between a specific module and the tissue type. T1, T2, T3, T4, and T5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of the microbial fertilizer treatment, CK1, CK2, CK3, CK4, and CK5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of the control treatment, respectively.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
GO enrichment analysis of each module highlighted key biological processes represented by a set of coexpressed genes and corroborated our results of differential gene expression analysis. For example, GO terms associated with modules of early-stage development (T1) were related to secondary metabolic processes, phenylpropanoid metabolic, secondary metabolite biosynthetic processes, and developmental processes (Supplemental Table 3). GO terms of the light cyan module associated with T4 were related to the sporopollenin biosynthetic process, pollen wall assembly, and cellular component assembly involved in morphogenesis. Likewise, GO terms of the modules associated with CK1 were related to the electron transport chain, photosynthetic electron transport in photosystem II, and generation of precursor metabolites and energy. GO terms of the brown module associated with CK4 included most genes involved in the polysaccharide metabolic process, response to oxygen-containing compound, and secondary metabolic process.
Differentially expressed transcription factor genes at different leaf stages.
Transcription factors play crucial roles in controlling plant growth, development, and phase changes by regulating gene expression (Mitsuda and Ohme-Takagi 2009; Riechmann et al. 2000). Multiple transcription factors were differentially expressed, including basic helix-loop-helix protein (bHLH), CCCH zinc finger proteins (CCCH), NAM/ATAF/CUC transcription factors (NAC), MYB transcription factors (MYB), WRKY transcription factors (WRKY), ethylene response factors (ERF), and RAX transcription factors (RAX) (Table 2, Fig. 4). Among them, 14 were identified at the four-leaf stage, including five upregulated MYBs, three upregulated and two downregulated bHLHs, one upregulated CCCH, one downregulated RAX and two downregulated NACs. Twenty-six genes were differentially expressed at the six-leaf stage, with more (17) downregulated genes, including one bHLH, one CCCH, two RAX1s, one MYC, four WRKYs, and eight ERFs, than upregulated genes. Twelve recognized differentially expressed transcription factors were identified at the eight-leaf stage, including one upregulated MYBs, two highly expressed NAC, one overexpressed MADS-box, and eight downregulated other genes. Nine and seven DEGs at 10- to 12-leaf stages were transcription factors, including one upregulated and two downregulated MYBs, three upregulated and one downregulated bHLHs, one upregulated CCCH, one upregulated and five downregulated NACs, and two upregulated ERFs.
Differentially expressed genes (DEGs) in inflorescences of ‘Summer Black’ grape at different leaf stages after fertilization.
Heatmap of differentially expressed genes associated with transcription factors in the inflorescence of ‘Summer Black’ grape treated with fertilization and control. T1, T2, T3, T4, and T5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of the microbial fertilizer treatment, CK1, CK2, CK3, CK4, and CK5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of the control treatment, respectively.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
DEGS related to phytohormone signaling at different leaf stages.
Plant growth, development, and interaction with the environment involve the action of multiple phytohormones. Phytohormones integrate endogenous and exogenous signals to synchronize plant growth with environmental and developmental changes (Borghi et al. 2015). A search of our DEGs data revealed 22 DEGs related to plant hormone signal transduction, including eight, eight, two, and four DEGs at the 4-, 6-, 10-, and 12- leaf stages, respectively (Table 3, Fig. 5). At the four-leaf stage, four genes were upregulated, and four genes were downregulated. At the six-leaf stage, one ARR gene and two cysteine-rich secretory protein family genes were upregulated, and one gibberellin receptor GID1A, two glycosyl hydrolases family 16, one protein TIFY, and one transcription factor MYC2 were downregulated. At the 10-leaf stage, one upregulated transcription factor bHLH 14 and one downregulated AUX/IAA family were associated with phytohormone signaling. At the 12-leaf stage, four downregulated DEGs (two cysteine-rich secretory protein families, one bZIP transcription factor, and one transcription factor bHLH14) were related to phytohormone signaling.
Differentially expressed genes associated with plant hormone signal transduction in inflorescence of ‘Summer Black’ grape at different leaf stages.
Heatmap of differentially expressed genes associated with phytohormone signal transduction in the inflorescence of ‘Summer Black’ grape treated with fertilization and control. T1, T2, T3, T4, and T5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of the microbial fertilizer treatment, CK1, CK2, CK3, CK4, and CK5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of the control treatment, respectively.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Validating gene expression patterns by qRT-PCR.
To validate further the expression patterns of phytohormone signaling-related genes revealed by RNA sequencing, qRT-PCR was conducted to examine the expression levels of six randomly selected DEGs at different leaf stages. As shown in Fig. 6, the results of qRT-PCR were completely consistent with the expression data obtained by RNA-seq.
Relative expression levels of six differentially expressed genes by quantitative real-time polymerase chain reaction. T1, T2, T3, T4, and T5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of ‘Summer Black’ grape microbial fertilizer treatment, CK1, CK2, CK3, CK4, and CK5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of ‘Summer Black’ grape control treatment, respectively.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Discussion
Inflorescence elongation, which can dramatically influence grape production, can be affected by microbial fertilizers. A systematic study of gene expression change is helpful in uncovering the molecular mechanism of changes in grape inflorescence elongation after applying fertilizers and provides valuable resources for improving grape production.
Inflorescence transcriptomes are rapidly and significantly regulated at different leaf stages by microbial fertilizers.
From the 4- to 12-leaf stages, inflorescences were gradually elongated, and hundreds of DEGs were found by comparing different groups at the same stage, indicating that microbial fertilizers may be responsible for the rapid and significant molecular changes in inflorescences. In the tea plant (Camellia sinensis), applying nitrogen fertilizers significantly increased leaf yield and affected the expression of 225 genes (Li et al. 2017). A previous study identified 81 and 78 DEGs under low and high nitrogen treatments, respectively (Ma et al. 2019). Therefore, fertilizers may be responsible for the rapid and significant changes in gene expression in ‘Summer Black’ grape inflorescences.
Starch and sucrose metabolism is activated in the microbial fertilizer group.
Sucrose metabolism is vital to fruit development. At different inflorescence elongation stages, many upregulated genes in the microbial fertilizer group were enriched in starch and sucrose metabolism (Supplemental Figs. 1–10). One hundred fifty-two unigenes were identified to be involved in starch and sucrose metabolism in the study of Chinese chestnut (Castanea mollissima) seed, and the expression of 21 unigenes putatively coding for major enzymes in starch and sucrose metabolism was validated by qPCR using RNAs collected from five seed stages (Zhang et al. 2015). The formation and development of onion (Allium cepa) bulbs were also closely related to sucrose metabolism (Zhang et al. 2016). The patterns of sucrose accumulation in yellow nut sedge (Cyperus esculentus) during tuber development were similar to those that occurred in developing sink organs for seeds of arabidopsis, rapeseed (Brassica napus), and tobacco (Nicotiana tabacum) (Yang et al. 2018). We found that sucrose synthase genes and starch phosphorylase genes were upregulated after applying microbial fertilizers. Similarly, the activities of soluble acid invertase, cell wall-bound invertase, sucrose synthase at cleavage direction, and starch phosphorylase were significantly increased in the rapidly elongating internodes of arrow bamboo (Fargesia yunnanensis). These enzymes dominated the rapid elongation of internodes (Wang et al. 2020). We speculated that many upregulated genes in the microbial fertilizer group were enriched in the starch and sucrose metabolism pathway and contributed to the increased starch and sucrose accumulation, an important reason for enhanced inflorescence elongation in the microbial fertilizer group.
Transcription factors are important regulators of inflorescence elongation.
Transcription factors that trigger major developmental decisions in plants are termed “master regulators.” Such master regulators are classically seen to act on the top of a regulatory hierarchy that determines a complete developmental program (Kaufmann and Airoldi 2018). For example, transcription factors are key regulators of wheat (Triticum aestivum) early spike development and involve in meristem maintenance (such as AP2 family transcription factors), flowering time modulation (e.g., TaSVP, AP1, and SOC1 homologs), regulation of meristem initiation or transition and floral organ development (e.g., WFL, TaLAX1, and Eps-3) (Li et al. 2018). In this study, multiple transcription factors, including bHLH, CCCH, NAC, MYB, WRKY, ERF, and RAX, were found to be differentially expressed between microbial fertilizer and control groups. Many differentially expressed transcription factors were identified at different leaf stages. For example, most bHLH, CCCH, and MYB were differentially expressed at all leaf stages (Table 2). Increasing reports have shown that CCCH zinc finger proteins are essential for plant development. AtC3H17, a unique arabidopsis gene encoding a nontandem CCCH zinc finger protein, was ubiquitously expressed throughout the life cycle of arabidopsis plants and their organs (Seok et al. 2016). A recent study has indicated that SlPRE2, a bHLH family transcription factor gene, is highly expressed in immature green fruit. SlPRE2 silencing reduced fruit size, pericarp thickness, and placenta size. In addition, SlPRE2-silenced fruit mesocarp had reduced cell size, and expression of SlXTH2 and SlXTH5 were involved in cell enlargement (Zhu et al. 2019).
MYB proteins, members of a large transcription factor family, are key regulators of the synthesis of phenylpropanoid-derived compounds and play critical roles in plant growth and development (Liu J et al. 2015). The expression levels of several bHLH, CCCH, and MYB were significantly altered by microbial fertilizer at every stage, indicating that bHLH, CCCH, and MYB play critical roles in regulating inflorescence elongation in ‘Summer Black’ grape.
Multiple phytohormone genes play pivotal roles in regulating inflorescence elongation.
Multiple phytohormones play important roles in plant growth and development. It is well known that gibberellin can control stem and internodal elongation by stimulating the degradation of nuclear growth-repressing DELLA proteins (Davière et al. 2014). Cytokinin is an important regulator in controlling inflorescence branching in Barbadosnut (Jatropha curcas; Chen MS et al. 2019). The content of GA3 was evidently higher in the microbial fertilizer group than in the corresponding CK group, indicating that applying microbial fertilizers effectively improved GA3 content in the leaves and inflorescence (Fig. 1). Meanwhile, our study revealed that 22 DEGs at different leaf stages were associated with phytohormones signal transduction. Gibberellin receptor GID1A, glycosyl hydrolases family 16, protein TIFY, MYC2, bHLH 14, AUX/IAA family, and b-ZIP transcription factor were differentially expressed and involved in the phytohormone pathway. Gibberellin controls higher plants’ growth and developmental processes (Murase et al. 2008). GID1A can perceive gibberellin and initiate a signaling cascade in the cytosol (Livne and Weiss 2014), which explains GID1A differential expression at the six-leaf stage. Auxin is an important hormone in the plant developmental process (Chen L et al. 2019). Functional analyses of AUX/IAA family members have indicated that they play various roles during plant development, such as root development, shoot growth, and fruit ripening (Luo et al. 2018), in many plants, including wheat (Qiao et al. 2017), bletilla (Bletilla striata; Liu H et al. 2019), dragon bamboo (Dendrocalamus sinicus; Chen L et al. 2019), and arabidopsis (Lakehal et al. 2019). However, the specific regulation mechanisms by which AUX interacts with inflorescence elongation in plants remain unclear.
Our transcriptomic profiling analysis of ‘Summer Black’ grape revealed dynamic gene expression changes in response to microbial fertilizers at different stages of inflorescence elongation. We found that applying microbial fertilizers effectively improved GA3 contents in ‘Summer Black’ grape leaves and inflorescence and activated starch and sucrose metabolism and revealed that multiple phytohormones genes play pivotal roles in regulating inflorescence elongation. These results provide valuable insight into the dynamic transcriptomic responses of inflorescence elongation at different leaf stages.
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Statistics of transcriptome sequencing data for each inflorescence sample of ‘Summer Black’ grape. T1, T2, T3, T4, and T5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of the microbial fertilizer treatment. CK1, CK2, CK3, CK4, and CK5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of the control treatment, respectively. Each treatment had three replicates.
Alignment results of each inflorescence sample of ‘Summer Black’ grape. T1, T2, T3, T4, and T5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of the microbial fertilizer treatment. CK1, CK2, CK3, CK4, and CK5 correspond to 4-, 6-, 8-, 10-, and 12-leaf stages of the control treatment, respectively. Each treatment had three replicates.
Key biological processes of Gene Ontology (GO) enrichment analysis in some coexpression network analysis modules of inflorescence transcriptome of ‘Summer Black’ grape.
Supplemental Fig. 1. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the upregulated differential genes at the 4-leaf stage of ‘Summer Black’ grape.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Supplemental Fig. 2. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the downregulated differential genes at the 4-leaf stage of ‘Summer Black’ grape.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Supplemental Fig. 3. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the upregulated differential genes at the 6-leaf stage of ‘Summer Black’ grape.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Supplemental Fig. 4. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the upregulated differential genes at the 8-leaf stage of ‘Summer Black’ grape.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Supplemental Fig. 5. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the upregulated differential genes at the 10-leaf stage of ‘Summer Black’ grape.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Supplemental Fig. 6. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the upregulated differential genes at the 12-leaf stage of ‘Summer Black’ grape.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Supplemental Fig. 7. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the downregulated differential genes at the 6-leaf stage of ‘Summer Black’ grape.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Supplemental Fig. 8. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the downregulated differential genes at the 8-leaf stage of ‘Summer Black’ grape.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Supplemental Fig. 9. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the downregulated differential genes at the 10-leaf stage of ‘Summer Black’ grape.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22
Supplemental Fig. 10. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the downregulated differential genes at the 12-leaf stage of ‘Summer Black’ grape.
Citation: Journal of the American Society for Horticultural Science 148, 4; 10.21273/JASHS05271-22