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Does temperature affect plant growth?


Contents

Introduction

Plants regulate their development in response to their surroundings. Temperature, in particular, is a significant element influencing plant development. Each plant species has a temperature range that is good for it. Higher temperatures within this range often encourage shoot development, including leaf expansion and stem elongation and thickness. Temperatures exceeding the ideal range, on the other hand, inhibit growth. Apart from the absolute temperature, the temperature variation between day and night may have an impact on development. Night temperatures are often lower than daytime temperatures, and plants respond to this temperature variation by modulating their growth pattern and metabolism. One approach of controlling plant development in decorative horticulture is to change the temperature differential between day and night. The difference in temperature between day and night, abbreviated as DIF, is defined as DT- NT. Positive DIF happens when DT exceeds NT, zero DIF occurs when DT equals NT, and negative DIF occurs when DT is less than NT. As an alternative to agricultural herbicides, negative DIF may be used to regulate plant height (Shimizu, 2007). Although the effects varied according to plant species, it has been documented that negative DIF reduces stem elongation more than positive DIF in a variety of plants, including Lilium longiflorum , Dendranthema grandiflora , Cucumis sativus (Shimizu, 2007), and Solanum lycopersicum (Went, 1944; de Koning, 1988).

Prior research has shown that phytohormones have a role in temperature-regulated plant development. Gibberellin (GA) has been shown to be important in stem elongation (Davière and Achard, 2013; Binenbaum et al., 2018; Ferrero et al., 2019). Indole-3-acetic acid (IAA), an auxin, is also essential for cell elongation in the hypocotyl, epicotyl, and other organs (Leyser, 2018; Zhao, 2018). In Arabidopsis thaliana Higher temperatures stimulate hypocotyl elongation through auxin production mediated by phytochrome-interacting factor 4 (PIF4) (Franklin et al., 2011; Nomoto et al., 2012; Sun et al., 2012). It has been demonstrated that PIF4 function is regulated by GA via DELLA proteins, which are key negative regulators of GA signaling (Koini et al., 2009; Stavang et al., 2009).

According to research, stem elongation under various DIF treatments is associated with a change in GA concentration in the stem. Campanula isophylla and Pisum sativum Jensen et al. (1996); Grindal et al. (1998); Stavang et al. (2005). In P. sativum , inhibition of stem elongation under negative DIF was weaker in GA-related mutants than that in the wild type (Grindal et al., 1998). In A. thaliana , non-bioactive GA29 content was lower under a negative DIF treatment than that under a positive DIF treatment, while IAA concentration was higher under a positive DIF treatment than that under a negative DIF treatment (Thingnaes et al., 2003). In Raphaus sativus L., stem elongation differences under DIF treatments followed a similar pattern to variations in IAA content (Hayata et al., 2001). These studies suggest the involvement of these hormones in the effect of DIF on stem elongation. Yet, these hormones and the expression of their genes have received little attention. Moreover, temperature influences not only stem elongation but also stem thickness; however, the impact of DIF on vascular development has yet to be fully described.

In this study, we aimed to elucidate the mechanisms underlying stem growth regulation by DIF. To that objective, we investigated the effects of several DIF treatments on tomato ( S. lycopersicum ), one of the world’s most significant vegetable crops. Our analyses of growth, transcriptomes, and hormones strongly suggest that negative DIF-dependent inhibition of stem elongation is mediated by the repression of GA and IAA synthesis accompanied by the regulation of cell wall-related genes. We also found that negative DIF therapy had little influence on tomato seedling stem thickening.

Results

Higher Temperatures Under Positive DIF Promote Stem Elongation and Thickening in Tomato Seedlings

To examine the effect of temperature under positive DIF on plant growth, tomato seedlings (Managua RZ) were grown under control (25°C/20°C, CT) and high temperatures (30°C/25°C, HT) (Supplementary Figures 1A,B). The lengths and diameters of the stem, hypocotyl, and epicotyl were substantially higher under HT than under CT (Figures 1A–C). The number of xylem vessels (diameter > 50 m) were considerably bigger under HT than under CT (Figures 1D,E), demonstrating that higher temperatures under positive DIF stimulate stem elongation, stem thickness, and vascular development in tomato seedlings.

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Figure 1. The effect of temperature on tomato seedling development. Young tomato seedlings were grown for 7 days under 25°C/20°C (control temperature) and 30°C/25°C (high temperature). (A) , Seedling development. At 7 days, tomato seedlings were photographed. The scale bar measures 5 cm. (B) , Hypocotyl and epicotyl length. (C) Diameters of the hypocotyl and epicotyl. Data are shown as boxplots ( n = 7). (D) , Hypocotyl cross sections. Asterisks denote xylems (diameters greater than 50 m). Scale bars = 500 m (left and center panels) and 50 m (right panel) (right panels). (E) , Number of xylems (diameter > 50 μm) in the cross section. Boxplots are used to display data ( n = 5). Cross marks in boxplot indicate the mean values. * P < 0.01; ** P < 0.001 (Student’s t CT stands for control temperature; HT stands for high temperature; HL stands for hypocotyl length; EL stands for epicotyl length; and X stands for xylem area.

Negative DIF Inhibits Stem Elongation, but Maintains Promotion of Stem Thickening

Tomato seedlings were cultivated under HT (30°C/25°C, positive DIF) or with night and day temperatures reversed (25°C/30°C, negative DIF) to investigate the impact of negative DIF on tomato seedling growth (Supplementary Figures 1B,C). Temperature-dependent stem elongation was reduced in both the hypocotyl and epicotyl under negative DIF therapy (Figures 2A,B). The difference in hypocotyl and epicotyl elongation between DIF treatments was detectable 3 days after treatment initiation and became significant at 5 days (Supplementary Figures 2A–C). In contrast, hypocotyl and epicotyl thicknesses were comparable across both DIF regimens (Figure 2C and Supplementary Figures 2D,E). The number of xylem vessels (diameter > 50 m) was likewise comparable (Figures 2D,E), demonstrating that the negative DIF treatment hindered stem extension without impairing stem thickness. The size of the cotyledons and genuine leaves was likewise comparable across treatments (Supplementary Figure 2F).

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Figure 2. The effect of DIF therapy on tomato seedling development. Young tomato seedlings were grown for 7 days under 30°C/25°C (positive DIF) and 25°C/30°C (negative DIF). (A) , Seedling development. Seedlings of tomato were photographed at 7 days. The scale bar measures 5 cm. (B) , Hypocotyl and epicotyl length. (C) Diameters of the hypocotyl and epicotyl. Data are shown as boxplots ( n = 5). (D) , Hypocotyl cross sections. Asterisks denote xylems (diameters greater than 50 m). Scale bars = 500 m (left and center panels) and 50 m (right panel) (right panels). (E) , The number of xylems in the cross section with diameters greater than 50 m. Data are shown as boxplots ( n = 5). The mean values are shown by the cross marks in the boxplot. P < 0.01; ** P < 0.001 (Student’s t +DIF denotes positive DIF; -DIF denotes negative DIF; HL denotes hypocotyl length; EL denotes epicotyl length; X denotes xylem area.

We examined the impact of the identical DIF treatments on four different tomato cultivars to see whether these responses were cultivar-specific. The negative DIF treatment, like Managua RZ, reduced stem length in all four tomato cultivars but had no effect on stem thickness (Supplementary Figure 3). This shows that the observed growth responses are frequent in tomato seedlings.

Negative DIF Affects Gene Expression

To examine the mechanisms underlying stem growth regulation under the DIF treatments, we analyzed epicotyl transcriptomes in seedlings grown under positive or negative DIF for 7 d (Supplementary Figures 1B,C) and explored differentially expressed genes (DEGs). Microarray research identified over 5000 DEGs, some of which were elevated and some of which were downregulated by negative DIF therapy (Figure 3A). We used the top 300 upregulated and downregulated genes to conduct gene ontology (GO) analysis ( P < 0.05) (Supplementary Tables 1, 2). (Supplementary Tables 1, 2). Enriched GO terms in biological process were found in cell wall macromolecule catabolic/metabolic process and (programmed) cell death (Figure 3B). Since stem elongation is linked to cell wall alteration, we concentrated our research on cell wall-related genes. In addition, hormone-related genes could be involved in temperature-dependent regulation of epicotyl elongation; consequently, GA and IAA-related genes were also included in further analyses. Seven DEGs related to the cell wall, GA, and IAA, as well as a PIF4 For further investigation, a homolog likely associated to temperature-dependent hypocotyl elongation was selected (Figure 3C).

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Figure 3. Tomato transcriptome study under positive and negative DIF treatments. Total RNA was extracted from epicotyls of tomato seedlings grown for 7 days under positive DIF and negative DIF and subjected to microarray analysis with three biological replicates. (A) MA plots of microarray data in positive and negative DIF treatments. In contrast to positive DIF, red and blue dots reflect genes that are elevated (Up: 5266 genes) and downregulated (Down: 5159 genes). Gray dots represent genes that did not significantly differ between DIF treatments. (B) , GO keywords enriched in DEGs (top 300 upregulated genes and top 300 downregulated genes; P < 0.05) identified. (C) , A list of eight genes linked to stem elongation. The relative expression level of each gene under negative DIF compared with that under positive DIF is shown on the heatmap, which represents the log2 fold-change (FC). The color scale may be seen at the bottom. +DIF, positive DIF; -DIF, negative DIF; BP, Biological Process; MF, Molecular Function.

GA and IAA Biosynthesis Genes Were Downregulated in Stems Under the Negative DIF Treatment

Among the identified DEGs, a key gene of de novo GA biosynthesis, namely GA20-oxidase ( SlGA20ox1 : Solyc03g006880), was suppressed (Figure 3C). Using reverse transcriptase real-time PCR, we confirmed that the expression of SlGA20ox1 was significantly downregulated in both hypocotyls and epicotyls under the negative DIF treatment (Figure 4A). Among the downregulated DEGs were the following IAA biosynthetic genes: YUCCA (YUC) encoding a flavin monooxygenase-like enzyme; TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) ; and IAA responsive factor SMALL AUXIN UP RNA (SAUR) (See Figure 3C). Our reverse transcriptase real-time PCR analysis confirmed that SlTAA1 (Solyc05g031600) and SlYUC (Solyc08g068160) were downregulated in tomato epicotyl tissues when treated with negative DIF (Figures 4B,C). SlSAUR47 After negative DIF therapy, (Solyc04g053000) was dramatically downregulated in both hypocotyl and epicotyl tissues (Figure 4D).

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Figure 4. Expression of GA-, IAA-, and cell wall-related genes in epicotyls and hypocotyls under negative and positive DIF treatments. The manifestation of SlGA20ox1 (A) , IAA-related genes ( SlTAA1 , SlYUC , SlSAUR47 ) (B–D) , cell wall-related genes ( SlEXP2 , SlEXP1 , SlXTH2 ) (E–G) , and SlPIF4 (H) in epicotyls and hypocotyls grown under positive and negative DIF treatments were analyzed using reverse transcriptase real-time PCR. Boxplots are used to display data ( n = 3). The mean values are shown by the cross marks in the boxplot. P < 0.1; ** P < 0.05; *** P < 0.01 (Student’s t test). +DIF, positive DIF; -DIF, negative DIF.

Cell Wall Modification Genes Were Downregulated in Stems Under the Negative DIF Treatment

In our transcriptome analysis, expansins ( SlEXP2 ; Solyc06g049050 and SlEXP1 ; Solyc06g051800) and Xyloglucan endotransglucosylase/hydrolase ( SlXTH2 ; Solyc07g009380) were suppressed (Figure 3C). Our reverse transcriptase real-time PCR analysis revealed that SlEXP2 and SlXTH2 With the negative DIF treatment, were considerably downregulated in both hypocotyls and epicotyls (Figures 4E,G). SlEXP1 After negative DIF therapy, was likewise downregulated in epicotyls (Figure 4F). These results are in line with the negative DIF treatment-driven repression of stem elongation.

We further examined the expression of PIF4 , but the expression of SlPIF4 (Solyc07g043580), a homolog of A. thaliana PIF4 , was not affected by DIF treatment (Figure 4H).

Higher Temperatures Under Positive DIF Upregulate Genes for GA and IAA Biosynthesis, as Well as Cell Wall Modification in Epicotyls

We next used reverse transcriptase real-time PCR to assess the expression of the DEGs under CT (25°C/20°C) and HT (30°C/25°C) positive DIF treatments (Supplementary Figures 1A,B). SlGA20ox1, SlYUC , SlEXP2 , and SlXTH2 were increased in epicotyls after HT therapy (Figures 5A,C,E,G). SlSAUR47 and SlEXP1 After HT treatment, were likewise elevated in both hypocotyls and epicotyls (Figures 5D,F). These findings support the favorable DIF treatment-driven stimulation of stem elongation. in addition to it, SlPIF4 was increased in epicotyls after HT therapy (Figure 5H).

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Figure 5. Expression of GA-, IAA-, and cell wall-related genes in epicotyls and hypocotyls under two different temperatures. The manifestation of SlGA20ox1 (A) , IAA-related genes ( SlTAA1 , SlYUC , SlSAUR47 ) (B–D) , cell wall-related genes ( SlEXP2 , SlEXP1 , SlXTH2 ) (E–G) , and SlPIF4 (H) in epicotyls and hypocotyls grown under control and high temperatures were analyzed by reverse transcriptase real-time PCR. Boxplots are used to display data ( n = 3). The mean values are shown by the cross marks in the boxplot. P < 0.1; ** P < 0.05; *** P < 0.01 (Student’s t CT stands for control temperature; HT stands for high temperature.

Phytohormone Concentrations Are Consistent With Stem Elongation and Gene Expression Patterns in Response to DIF Treatments

The influence of growing temperature on phytohormone concentrations in hypocotyls and epicotyls was also examined (Figure 6). GA1 and GA4, bioactive forms, could only be measured in epicotyls with positive DIF therapy, but they were below the quantification limit in all other tissues under negative DIF treatment (Figures 6A,B). GA7, another bioactive form, was likewise found to be below the quantitative limit (Figure 6C). The concentrations of the precursors, including GA9, GA19, GA20, GA24, and GA44, were lower in hypocotyls or epicotyls, while that of GA53 was higher, under the negative DIF treatment compared with those under the positive DIF treatment (Figures 6D,F–J).

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Figure 6. GA and IAA concentration in hypocotyls and epicotyls under negative and positive DIF treatments. Hypocotyls and epicotyls of tomato seedlings grown for 7 days under positive and negative DIF treatments were harvested and subjected to hormone analysis. The GA concentrations (A–J) and IAA (K) were quantified using liquid chromatography-tandem mass spectrometry. The data is displayed as mean SD ( n = 4 or 5). * P < 0.1; ** P < 0.05; *** P < 0.01 (Student’s t test). GA7 and GA12 fell below the quantitation threshold. +DIF, positive DIF; -DIF, negative DIF; FW, fresh weight; UQ, under quantification limit.

When we analyzed hormone species under the CT and HT positive DIF treatments (Figure 7), GA1 and GA4 were only detected in epicotyls under the HT treatment (Figures 7A,B). Concentrations of the inactive precursors GA15, GA24, and GA44 in epicotyls were higher under the HT treatment than those under the CT treatment (Figures 7E,H,I). In contrast, GA53 concentrations were lower in the HT treatment than in the CT therapy (Figure 7J). These patterns were diametrically opposed to the alterations seen after negative DIF therapy (Figure 6).

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Figure 7. The concentrations of GA and IAA in hypocotyls and epicotyls were measured at two different temperatures. Hypocotyls and epicotyls of tomato seedlings grown for 7 days under control and high temperatures were harvested and subjected to hormone analysis. The GA concentrations (A–J) and IAA (K) were quantified using liquid chromatography-tandem mass spectrometry. The data is displayed as mean SD ( n = 4 or 5). * P < 0.1; *** P < 0.01 (Student’s t test). GA7 and GA12 fell below the quantitation threshold. CT, control temperature; HT, high temperature; FW, fresh weight; UQ, under quantification limit.

IAA concentrations in negative DIF-treated hypocotyls and epicotyls were slightly but significantly lower than those in positive DIF-treated tissues (Figure 6K). In contrast, the concentrations of IAA in hypocotyls and epicotyls were higher under HT than those under CT (Figure 7K). These findings matched the expression patterns of IAA production and signaling genes (Figures 4, 5).

We also quantified the concentration of cytokinins (CKs) because the involvement of this hormone in vascular development is well-documented (Kieber and Schaller, 2014). Several N 6-(Δ2-isopentenyl) adenine (iP)-type and trans -zeatin-type (tZ-type) species had higher concentrations in negative DIF-treated epicotyls than positive DIF-treated epicotyls (Supplementary Figure 4). The CT and HT treatments, on the other hand, had the opposite impact (Supplementary Figure 5).

Discussion

In this study, we demonstrated that DT and NT affect stem growth in tomato seedlings, and that this is possibly mediated by the regulation of GA-, IAA-, and cell wall-related genes. Higher temperatures increased stem elongation in the positive DIF treatment, which was followed by an overexpression of GA and IAA synthesis genes, leading in larger concentrations of their active forms. Negative DIF, on the other hand, reduced stem elongation by downregulating GA and IAA synthesis genes and decreasing GA and IAA concentrations. Despite the fact that many research have been conducted to examine plant growth control in response to temperature, our work gives information regarding potential mechanisms governing stem development under DIF treatments.

Under the negative DIF treatment, the concentrations of GA1, GA4, and some GA precursors were lower, whereas those of GA53 were higher, compared with those under positive DIF treatment. Additionally, the manifestation of SlGA20ox1 was suppressed by the negative DIF therapy. In contrast, under the positive DIF treatment (i.e., higher temperatures), upregulation of SlGA20ox1 was observed and this treatment had the opposite effect on the profile of GA1, GA4 and the precursors, compared to that of the negative DIF treatment. Since GA20ox catalyzes the multi-step processes listed below: GA12/GA53→GA15/GA44→GA24/GA19→GA9/GA20, these results were consistent with the downregulation or upregulation of SlGA20ox1 expression. In addition, as GA8 is the inactivated form of GA1, lower GA8 concentrations in negative DIF-treated epicotyls supported weakened GA activity. GA20ox plays a key role in the GA biosynthesis pathway and affects bioactive GA content (Yamaguchi, 2008). In A. thaliana , the over-expression of GA20ox increases hypocotyl elongation and the expression of (Huang et al., 1998; Coles et al., 1999; Ferrero et al., 2019). GA20ox ( GA20ox1 ) is upregulated by high temperatures in hypocotyls (Stavang et al., 2009). As a result, it is argued that SlGA20ox1 regulates stem elongation in tomato seedlings in response to diverse DIF conditions.

IAA is involved in thermomorphogenesis, such as stem elongation, in response to higher temperatures (Quint et al., 2016). In A. thaliana High temperatures stimulate the expression of hypocotyls. YUC , TAA and SAUR , which promotes stem elongation (Stavang et al., 2009; Franklin et al., 2011). In our analysis, these homologs were downregulated under the negative DIF treatment and upregulated under the high temperature positive DIF treatment, suggesting that these IAA-related genes also play a role in regulating stem elongation in tomato in response to temperature conditions.

Expression patterns of the cell wall-related genes SlEXP1 , SlEXP2 , and SlXTH2 were linked to genes involved in GA and IAA production. EXPs are proteins that loosen the cell wall and cause it to expand (Marowa et al., 2016). Past research has shown that EXPs are involved in stem elongation of Oryza sativa and react to GA (Cho and Kende, 1997a,b; Lee and Kende, 2001, 2002; Choi et al., 2003; Zou et al., 2015). (Cho and Kende, 1997b; Lee and Kende, 2001, 2002). EXP1 has been found to be regulated by temperature in Agrostis scabra and Agrostis stolonifera 2007; Xu et al. XTH catalyzes Xyloglucan endohydrolysis and endotransglycosylation, which is involved in the modification of cell wall structures (Rose et al., 2002). Also, it has been stated that EXPs and XTHs are governed by the IAA (Goda et al., 2004; Majda and Robert, 2018; Lehman and Sanguinet, 2019). The response of cell wall-related genes to temperature might be mediated by GA and/or IAA action in tomato seedlings.

Prior research examined the molecular pathways underlying temperature acclimatization in A. thaliana identified PIF4 as a key regulator (Proveniers and van Zanten, 2013; de Wit et al., 2014; Quint et al., 2016). Manifestation of PIF4 was upregulated by high temperatures and was found to control GA and IAA biosynthesis and signaling (Koini et al., 2009; Stavang et al., 2009; Franklin et al., 2011; Sun et al., 2012). In our investigation, SlPIF4 was upregulated in epicotyls under the high temperature positive DIF treatment, suggesting that similar regulatory systems are employed in tomato seedlings. Conversely, SlPIF4 The negative DIF therapy had no effect on expression. The function of PIF4 in stem elongation control under negative DIF remains unknown. Future research should concentrate on creating a loss-of-function mutant to better understand its function.

Stem thickness and vessel development did not differ significantly between the negative and positive DIF treatments. Limited studies have reported the effect of temperature on stem thickness and vessel development. CKs are phytohormones that are vital in vascular formation (Kieber and Schaller, 2014). In a mutation of cytokinin biosynthesis, A. thaliana , stem thickness, and the quantity of xylems were all reduced dramatically (Matsumoto-Kitano et al., 2008). Another recent research reveals that CK is involved in xylem development variance in Dutch and Japanese tomato cultivars (Qi et al., 2020). However, in our experimental conditions, the concentration of endogenous cytokinin was increased in the negative DIF and decreased in the positive DIF treatments, suggesting that CK plays a minor role in the regulation of xylem development under these DIF treatments. Further research is required to understand the processes that underpin temperature-dependent control of stem thickness and xylem development.

It is considered that the quality of seedlings has a significant impact on agricultural productivity. Our findings demonstrate that a short-term negative DIF therapy may limit plant height without changing stem thickness. This might be a good method for cultivating tomatoes in nurseries. Since stem thickening is accompanied by vascular development, it may give a positive effect on mineral transport, the partition of assimilates, and fruit growth. The study’s findings will give information on the processes of potential new farming methods.

Materials and Methods

Plant Materials and Growth Conditions

The tomato ( S . lycopersicum Except for the growth comparison of tomato cultivars under DIF treatments, the cultivar employed in this research was Managua RZ (RIJK ZWAAN, Netherlands) (Supplementary Figure 3). In the comparison experiment, we used CF Momotaro-York and Daiki B Baria (Takii Seed, Kyoto, Japan), and Rinka and Reiyo (Sakata Seed, Kanagawa, Japan).

Tomato seeds were immersed in water for two days in a petri dish at 28°C in the dark. Following imbibition, germinated seeds were placed to a wet rockwool block (Nippon Rockwool Company, Japan) and cultivated for 5-6 days at 23°C/23°C (DT/NT, 16 h photoperiod) under fluorescent lighting of 130 to 140 mol photons m-2 s-1. Next, the young seedlings, whose cotyledons were fully opened, were further grown on the rockwool block with liquid culture medium under the following two temperature-condition pairs: 25°C/20°C and 30°C/25°C, or 30°C/25°C and 25°C/30°C (DT/NT, 16 h photoperiod) under 300 μmol photons m–2 s–1 of light for 7 d (Supplementary Figure S1). The liquid culture media contains the following nutrients: 5 mM KNO3, 1 mM NH4H2PO4, 0.5 mM MgSO4, and 5.5 mM Ca (NO3) 2, 27M Fe-EDTA, 25M KCl, 10M H3BO3, 1M MnSO4, 1M ZnSO4, 0.25M CuSO4, and 0.04M Na2MoO4.

Fluorescent Observation of Xylem

The tomato seedling hypocotyls were removed and treated in a 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque, Kyoto, Japan). The preserved hypocotyls were thinly sliced (0.5 mm) using a razor blade, and fluorescence microscopy was used to examine cross sections (Mirror unit with U-FUW, Olympus BX53, Olympus, Japan). The ImageJ program was used to measure the diameter of the vessels (Abramoff et al., 2004).

GA, IAA, and CK Quantification

Phytohormones were isolated and semi-purified in the same manner as previously reported (Kojima et al., 2009; Kojima and Sakakibara, 2012). CKs were quantified using an ultra-performance liquid chromatography (UPLC)-electrospray interface (ESI) tandem quadrupole mass spectrometer (qMS/MS) (AQUITY UPLCTM System/Xevo-TQS; Waters, Milford, MA, United States) as described previously (Kojima et al., 2009) with an ODS column (AQUITY UPLC HSS T3, 1.8mm, 2.1 x 100 mm; Waters). IAA and GAs were quantified using an ultra-high performance liquid chromatography (UHPLC)-ESI quadrupole-orbitrap mass spectrometer (UHPLC/Q-ExactiveTM; Thermo Fisher Scientific, United States) as described previously (Kojima and Sakakibara, 2012; Shinozaki et al., 2015) with an ODS column (AQUITY UPLC HSS T3, 1.8mm, 2.1 x 100 mm; Waters).

RNA Extraction

Using a mortar and pestle, hypocotyls and epicotyls were frozen in liquid nitrogen and crushed to a fine powder. Total RNA was extracted using an RNeasy Mini kit with an RNase-Free DNase Set (Qiagen, Hilden, Germany, Cat. No. 74104/79254). A NanoDrop-1000 spectrophotometer was used to quantify the RNA in the samples, and an Agilent 2100 Bioanalyzer was used to evaluate the quality (Agilent Technologies, Santa Clara, CA, United States).

Microarray and Data Analysis

Total RNA was extracted from epicotyls of young seedlings grown for 7 d under 30°C/25°C (positive DIF) and 25°C/30°C (negative DIF) (DT/NT, 16 h photoperiod) as described in the preceding subsection. For microarray analysis, three biological replicates were employed. Target labeling was carried out in accordance with the Low Input Rapid Amp Labeling Kit, One-Color instructions (Agilent Technologies). We utilized a custom-designed tomato microarray (platform ID “GPL21511”). The hybridization was carried out in accordance with the manufacturer’s instructions. We used an Agilent DNA Microarray Scanner G2565CA to scan the microarray pictures (Agilent Technologies). Feature Extraction software was used to convert scanned pictures to signal data (Agilent Technologies). The data matrix’s value definition was Log2. GO enrichment analysis among DEGs (top 300 upregulated and top 300 downregulated genes; P The GO Analysis Toolkit and Database for Agricultural Communities (AgriGO, http://systemsbiology.cau.edu.cn/agriGOv2/) was used (Tian et al., 2017).

Reverse Transcriptase Real-time PCR

SuperScriptTM III First-Strand Synthesis SuperMix was used to create first-strand cDNA (Invitrogen, Waltham, MA, United States). Real-time PCR was performed using a StepOnePlus Real Time PCR system (Applied Biosystems, Waltham, MA, United States) and a KAPA SYBR FAST qPCR Master Mix (2×) Kit (Kapa Biosystems, London, United Kingdom) under the following conditions: 95°C for 3 minutes, then 40 cycles of 95°C for 3 seconds and 60°C for 20 seconds. The CT technique was used to quantify gene expression, which was then normalized to that of the ubiquitin homolog as the housekeeping gene (Solyc01g056940). The following primers were used: for the housekeeping gene (Solyc01g056940), forward primer 5′-CG TGGTGGTGCTAAGAAGAG-3′, reverse primer 5′-ACGAAG CCTCTGAACCTTTC-3′; for SlGA20ox1 (Solyc03g006880), forward primer 5′-TGGCGTTCCATCAGTCCAAA-3′, reverse primer 5′-TTCGAGGGTTGTTGGAGTCC-3′; for further information, see SlTAA1 (Solyc05g031600), forward primer 5′-TGAAGCACACCCTGC ATTTG-3′, reverse primer 5′-ACTTCCAAATCTTTCCACT CCTT-3′; SlYUC (Solyc08g068160), forward primer 5′-GC CCTCGTGGCTAAAGGAA-3′, reverse primer 5′-CCACTGCA TAAAGTCCACACTCTC-3′ SlSAUR47 (Solyc04g053000), forward primer 5′-GAAGAACAGTTTGGCTTCGATTAC-3′, reverse primer 5′-CGGTATGTGATCAACAAACAAACAAACAAACAAACAAACAAACAAACAAACAAACAAACAAACAAACAAACAAACAAACAA -3′; for SlEXP2 (Solyc06g049050), reverse primer 5′-TGAATATCACCAGCAC CTCCA-3′, forward primer 5′-TTCGAAGGGTG CCCTGTAT-3′; for SlEXP1 (Solyc06g051800), forward primer 5′-CGCTGGCATTGTTCCTGT-3′, reverse primer 5′-CTGC ACCTGCTACATTCGTG-3′; for further information, see SlXTH2 (Solyc07g009380), forward primer 5′-TATGCACAAGGCAAGGGAGA-3′, reverse primer 5′-TGTATTGTCTTATTGGTGTCCATC-3′ SlPIF4 (Solyc07g043580), forward primer 5′-ATCAAGCAGCTGCAAT GTGC-3′, reverse primer 5′-CTGCTGAGTTTTGCTG-3′.

Data Availability Statement

The microarray data has been placed in the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) database with accession number GSE131496.

Author Contributions

The study was designed by KO, AY, SI, and HS. KO, AY, YK, KF, MK, YT, and KY performed research. The data was evaluated by KO and YK. The manuscript was written by KO and HS. The essay was written by all of the writers, and the final version was approved by all of them.

Funding

The Cross-ministerial Strategic Innovation Promotion Program (SIP) and JSPS KAKENHI Grant Number JP19H05462 helped fund this study.

Conflict of Interest

The authors state that no commercial or financial ties that might be considered as a possible conflict of interest existed during the study.

Acknowledgments

We thank Momo Okumura, Mayumi Tanigawa, and Mizuki Yamada for their advice on tomato cultivation. The National Agricultural and Food Research Organization participated with the microarray analysis (NARO).

Supplementary Material

This article’s Supplemental Material is available online at: https://www.frontiersin.org/articles/10.3389/fpls.2020.577235/full#supplementary-material

Supplementary Figure 1 | The study’s growing circumstances are shown schematically. (A) Control temperature treatment, 25°C/20°C (DT/NT). (B) 30°C/25°C (DT/NT) high temperature/positive DIF therapy. (C) Treatment with negative DIF at 25°C/30°C (DT/NT). In each environment, young tomato seedlings were cultivated for 7 days. White bars show the light period, black bars show the dark period, and red arrowheads show the sampling time for the experiments. Further information may be found in the Materials and Methods section.

Supplementary Figure 2 | Tomato plant growth under negative and positive DIF treatments. Young tomato seedlings were grown for 7 days under positive and negative DIF treatments. (A) The development of seedlings. Tomato seedlings were photographed at 0, 1, 3, 5, and 7 days. 5 cm scale bars ( B,C ) Hypocotyl and epicotyl length, respectively. ( D,E ) Hypocotyl and epicotyl diameters, respectively. (F) Cotyledons and true leaves photographed at 7 days after onset of DIF treatments. The scale bars are 5 cm long. The data is displayed as mean SD ( n = 3 or 4). ∗ P < 0.05; ∗∗ P < 0.01 (Student’s t test). +DIF, positive DIF; -DIF, negative DIF.

Supplementary Figure 3 | DIF treatments were used to compare the growth of five tomato varieties. Seedlings of five tomato cultivars, Daiki B baria (DaikiB), Rinka, CF momotaro-york (CFMY), Managua RZ (MA), and Reiyo (RYO) were grown for 7 days under positive and negative DIF. (A) The development of seedlings. Tomato seedlings were photographed after 7 days. 5 cm scale bars ( B,C ) Hypocotyl and epicotyl length, respectively. ( D,E ) Hypocotyl and epicotyl diameter, respectively. Boxplots are used to display data ( n = 3). Cross marks in boxplot indicate the mean values. ∗ P < 0.05; ∗∗ P < 0.01 (Student’s t test). +DIF, positive DIF; -DIF, negative DIF.

Supplementary Figure 4 | Concentration of CKs in hypocotyls and epicotyls under positive and negative DIF treatments. Tomato seedling hypocotyls and epicotyls were collected and hormone analysis was performed on hypocotyls and epicotyls grown for 7 days under positive and negative DIF treatments. Liquid chromatography-tandem mass spectrometry was used to determine CK concentrations. The data is displayed as mean SD ( n = 3 to 5). tZ, trans tZR, tZ riboside; tZRPs, tZR 5′-phosphates; tZ7G, tZ-zeatin 7N -glucoside; tZ9G, tZ- 9N -glucoside; tZOG, tZ- O -glucoside; tZROG, tZR- O -glucoside; iP, N 6-( Δ2 -isopentenyl)adenine; iPR, iP riboside; iPRPs, iPR 5′-phosphates; iP7G, iP-isopentenyl)adenine 7N -glucoside; iP9G, iP- 9N -glucoside. ∗ P < 0.1; ∗∗ P < 0.05; ∗∗∗ P < 0.01 (Student’s t test). tZRPOG, tZRP- O -glucoside was below the quantification limit. +DIF denotes positive DIF; -DIF denotes negative DIF; FW denotes fresh weight; UQ denotes under quantification limit.

Supplementary Figure 5 | Concentration of CKs in hypocotyls and epicotyls under two different temperatures. Hormone analysis was performed on hypocotyls and epicotyls of tomato seedlings cultivated for 7 days under control and high temperatures. Liquid chromatography-tandem mass spectrometry was used to determine CK concentrations. The data is displayed as mean SD ( n = 3 to 5). tZ, trans tZR, tZ riboside; tZRPs, tZR 5′-phosphates; tZ7G, tZ-zeatin 7N -glucoside; tZ9G, tZ- 9N -glucoside; tZOG, tZ- O -glucoside; tZROG, tZR- O -glucoside; iP, N 6-( Δ2 -isopentenyl)adenine; iPR, iP riboside; iPRPs, iPR 5′-phosphates; iP7G, iP-isopentenyl)adenine 7N -glucoside; iP9G, iP- 9N -glucoside. ∗ P < 0.1; ∗∗ P < 0.05; ∗∗∗ P < 0.01 (Student’s t test). tZRPOG, tZRP- O The concentration of -glucoside was below the quantitative limit. CT, control temperature; HT, high temperature; FW, fresh weight; UQ, under quantification limit.

Abbreviations

CT is for control temperature; DT stands for day temperature; +DIF stands for positive DIF; -DIF stands for negative DIF; HT stands for high temperature; and NT stands for night temperature.

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Related Questions

  • How does cold temperature affect plant growth?

    Cold temperatures may have a variety of effects on plants. For starters, temperatures around the lowest for plant development slow the plant’s metabolism and growth. If the temperature, and therefore the metabolism, remain low for an extended period, plant quality will suffer, and death may occur.

  • How does heat affect plant growth?

    Studies have shown that high temperatures can increase the plant’s rate of reproductive development, which shortens the time for photosynthesis to contribute to fruit or seed production. Heat stress issues make the plant more vulnerable to pests and other environmental issues.

  • Does high temperature affect plants?

    High temperatures are detrimental to the development of many plant species because the rate of photosynthesis (the fundamental mechanism by which plants produce sugar) tends to fall fast once a crucial high temperature is achieved.

  • What temperature is best for plant growth?

    70°F to 75°F

    To minimize overly extended production durations, plants should be cultivated at warm temperatures ranging from 70°F to 75°F. By growing cold-sensitive crops at warm temperatures, you can actually reduce the amount of energy used for heating — on a per-crop basis — than if they were grown at cooler temperatures.

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