Background: Lycopene is an important carotenoid pigment in red fruits and vegetables, especially in tomato. Although lycopene biosynthesis and catabolism have been found to be regulated by multiple factors including phytohormones, little is known about their regulatory mechanism. Cytokinins are crucial to various aspects of plant growth. Isopentenyltransferases (IPTs) catalyze the initial rate-limiting step of cytokinins biosynthesis, however, their roles in fruit ripening remain unclear. Results: Here, the functions of SlIPT4, encoding an isopentenyltransferase, were characterized via RNAi-mediated gene silencing in tomato. As we expected, silencing of SlIPT4 expression resulted in accelerated leaf senescence. However, down-expression of SlIPT4 generated never-red orange fruits, corresponding with a dramatic reduction of lycopene. Among lycopene biosynthesis-related genes, the fact of remarkable decrease of ZISO transcript and upregulation of other genes, revealed that SlIPT4 regulates positively lycopene biosynthesis via directly affecting ZISO expression, and also supported the existence of regulatory loops in lycopene biosynthesis pathway. Meanwhile, the accumulation of abscisic acid (ABA) was reduced and the transcripts PSY1 were increased in SlIPT4-RNAi fruits, supporting the feedback regulation between ABA and lycopene biosynthesis. Conclusion: The study revealed the crucial roles of SlIPT4 in leaf senescence and the regulatory network of lycopene biosynthesis in tomato, providing a new light on the lycopene biosynthesis and fruit ripening. Keywords: Carotenoids, Cytokinins, Fruit ripening, Leaf senescence, Lycopene, SlIPT4,Tomato Background Moreover, Lycopene is of particular nutritional interest in Carotenoids are a group of terpenoid pigments, naturally promoting health and reducing the risk of various dis- synthesized in plants, fungi, algae and photosynthetic bac- eases, especially cancer and cardiovascular disease [7–9]. teria , and usually give bright colors to fruit, flower and In recent years, significant progress has been achieved in seed in plants [2, 3]. Carotenoids are more than just pig- understanding of carotenoid biosynthesis and catabolism ments, they also act as membrane stabilizers and the pre- using biochemical and genetics approaches . Lycopene cursors of important plant hormones, such as abscisic is produced from carotenoid biosynthesis pathway, and has acid (ABA) and strigolactone, to play important roles in been proposed to proceed through a poly-cis pathway: ger- photosynthesis and a variety of physiological processes in- anylgeranyl diphosphate (GGPP)→ 15-cis-phytoene → cluding plant growth, fruit development and response to 9,15,9′-tri-cis-ζ-carotene → 9,9′-di-cis-ζ-carotene → proly- abiotic stress [4–6]. Lycopene is a bright red linear carot- copene → all-trans-lycopene, catalyzed by phytoene syn- enoid and widely exists in red fruits and vegetables. thase (PSY), phytoene desaturase (PDS), ζ-carotene isomerase (ZISO), ζ-carotene desaturase (ZDS), and caro- tene isomerase (CrtISO), respectively [2, 3, 10–12]. Lyco- * Correspondence: email@example.com pene can be further cyclized to produce carotenoids with Bioengineering College, Chongqing University, Chongqing 400044, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. BMC Plant Biology (2018) 18:107 Page 2 of 12 two β rings (β-carotene) and one β ring and one ε ring [25–28]. Throughout fruit ontogeny, it is known that CKs (α-carotene). In higher plants, lycopene-β-cyclase (LCYB) and their biosynthesis-related genes may play important and lycopene ε-cyclase (LCYE) catalyze the formation of roles in fruit set and development, however, to our know- β-rings and ε-rings, respectively [13, 14]. The α-carotene ledge, little is known about their impact on fruit ripening. and β-carotene can be hydroxylated to generate xantho- The adenosine phosphate-isopentenyltransferases (IPTs) phyll pigments lutein and zeaxanthin, respectively. Zeaxan- catalyze the initial step of CK biosynthesis to produce iso- thin is the precursor of apocarotenoid hormone ABA . pentenyladenine (ip) nucleotides as CK precursors, it is Plant carotenoid biosynthesis and catabolism have been the rate-limiting biosynthetic step of CKs [29, 30]. Plant found to be regulated by developmental programs, envir- IPTs belong to multigenic family and have been identified onmental factors, metabolic signals, and classic phytohor- in Arabidopsis (AtIPT1-AtIPT9) and tomato (SlIPT1-- mones including ethylene, auxin, and ABA [2, 6]. SlIPT6). Up to now, about the physiological signifi- Tomato (Solanum lycopersicum) is by far the largest cance of the six tomato SlIPTs,only SlIPT3 and SlIPT4 dietary source of lycopene , and also a model system were reported to mediate salt stress response in tomato for fleshy fruit development and ripening, exhibiting active . However, the authors only obtained the SlIPT3 over- cell division and expansion at the early developmental expression tomato lines, and didn’t generate any trans- stages, and dramatic changes in texture and carotenoid, genic tomato plants of SlIPT4 overexpression or sugar, and acid content during ripening stages . In to- knockdown. So the physiological function of tomato mato fruit, lycopene is the predominant carotenoid pig- SlIPT4 is still unknown. ment and principally responsible for the deep-red color, In the present study, we report that the tomato ISO- which is the most obvious characteristic of ripe fruit . PENTENYLTRANSFERASE4 gene (SlIPT4, GenBank ac- For lycopene accumulation during tomato fruit ripening, cession number AB690814), is involved in leaf senescence the transcripts of genes modulating lycopene biosynthesis and pigment formation of ripe fruit. RNAi-mediated silen- are upregulated, on the contrary, the expression of genes cing of SlIPT4 accelerated leaf senescence and generated encoding the enzymes that metabolize lycopene are dra- orange fruits with a significant reduction of lycopene con- matically decreased . Two tomato mutants altering tent. The dramatic reduction of ZISO transcripts, lycopene content have been characterized: yellow-flesh (locus moderate decrease of ZDS mRNAs, and upregulation r) exhibits yellow fruits as a result of a loss-of-function mu- of other genes (PSY1, PDS, CrtISOs)in SlIPT4-si- tant of the PSY1 gene , and tangerine mutant (locus t) lenced fruits, suggest that SlIPT4 controls lycopene appearsorangefruitsdue to amutationinthe CrtISO gene biosynthesis via directly affecting ZISO expression, . Additionally, several transcription factors and genes are and also support the existence of regulatory loops in involved in the regulatory network of carotenoid biosyn- the carotenoids biosynthesis pathway. These data un- thesis. The ripening inhibitor (RIN), a MADS box transcrip- cover that SlIPT4 is involved in the regulatory net- tion factor, is able to affect the accumulation of lycopene by work of lycopene biosynthesis and plays a crucial role directly interacting with the promoter of PSY1 gene . A in color formation during fruit ripening in tomato. STAY-GREEN protein SGR1 can regulate lycopene accumu- lation through directly interacting with PSY1 to inhibit its activity during tomato fruit maturation . RNAi-mediated Methods silencing of AP2a, encoding a tomato APETALA2/Ethylene Plant materials and growth condition Responsive Factor (AP2/ERF) transcription factor, re- Tomato (Solanum lycopersicum cv. MicroTom) plants duced total carotenoids accumulation and downregu- were grown under the following conditions: 14/10 h day/ lated the expression of PSY1 . In Arabidopsis,the night cycle, 25 °C/20 °C day/night temperature, 80% hu- − 2 − 1 AtAP2 and phytochrome-interacting factor 1 (AtPIF1) midity, and 250 μmol m s light intensity. For analyz- regulate carotenoid biosynthesis by directly binding to ing the organ-specific expression profiling of SlIPT4,the the promoter of AtPSY1 gene [23, 24]. Although these roots, stems, leaves, flowers, and fruits (mixture of mature genes were reported to regulate carotenoid biosyn- green stage) were collected from 8-week-old wild-type thesis, they focused mainly on repressing the PSY1 tomato plants. Samples taken from the different parts expression in plants. The knowledge on the exact (ovary, stamen, petal, and sepal) of flowers were har- regulation mechanism of carotenoid/lycopene biosyn- vested at bud (− 2 dpa, day post anthesis), anthesis (0 thesis in response to various developmental and en- dpa), and post-anthesis (4 dpa) stages, respectively. vironmental factors and so on is still limit so far. The developmental stages of fruits investigated in this The phytohormone cytokinins (CKs) play crucial roles in study were early mature green (25 dpa), mature green a wide aspects of plant growth and development, including (35 dpa), break (Br, the first visible sign of carotenoid cell division, fruit development, leaf senescence, apical accumulation is evident in surface, 40 dpa), orange dominance, lateral root formation, and stress tolerance (Br+3 days), and ripening (Br+7 days). Zhang et al. BMC Plant Biology (2018) 18:107 Page 3 of 12 Quantitative real-time PCR Chlorophyll and carotenoids measurements Total RNA samples were extracted using Trizol reagent Chlorophyll and carotenoids were measured as de- (Invitrogen, USA) according to the manufacturer’sinstruc- scribed by Forth and Pyke . Briefly, total chloro- tions. The first-strand cDNA synthesis was performed using phyll from 2 g of fresh expanded leaves and total 1 μg of total RNA by PrimeScript™ RT reagent Kit with carotenoids from 3 g of fresh ripe fruits were ex- gDNA Eraser (Takara, Japan). Quantitative real-time PCR tracted using hexane/acetone (3:2, v/v) and acetone/ (qRT-PCR) was performed using cDNAs corresponding to petroleum ether (1:4, v/v), respectively. After centrifu- 2ngoftotal RNA in 10 μL reaction volume using SYBR gation, the supernatant was measured using spectro- GREEN PCR Master Mix on a CFX96 Touch™ Real-Time photometer (PerkinElmer, USA). The amount was PCR Detection System (Bio-Rad, USA). The qRT-PCR reac- calculated with the following equations: total chloro- − 1 tions were performed as follow: 95 °C for 2 min, followed by phyll mg mL =8.02 (OD ) + 20.2 (OD ), and 643 647 − 1 40 cycle of 95 °C for 15 s and 58 °C for 40 s and one cycle total carotenoids mg mL =(OD )/0.25. -ΔΔCt of 95 °C for 15 s and 60 °C for 15 s. The 2 method was For quantification of lycopene, β-carotene and lutein, used for the analysis of relative gene expression levels as de- pigments were extracted from 2 g of fresh ripe fruits scribed by Yang et al. . For all qRT-PCR experiments, at using acetone/petroleum ether (1:1, v/v), then dried least three biological replicates were performed and each re- under a stream of N and dissolved in 100% dichloro- action was run in triplicate. Tomato Slactin-51 (GenBank methane. The HPLC analysis was performed with 10 μL accession number Q96483) was used as the reference gene dichloromethane-dissolved pigments on ACQUITY . The sequences of gene primers for qRT-PCR were UPLC (Waters, USA). Lycopene, β-carotene and lutein listed in Additional file 1:Table S1. were identified by their characteristic absorption spectra (472 nm, 450 nm and 446 nm, respectively) and distinct- RNA interference (RNAi) vector construction and plant ive retention times, compared with their corresponding transformation standard substance (Sigma, USA). Each carotenoid con- A 340 bp specific fragment of SlIPT4 was amplified tent was calculated through the linear regression equa- from tomato cDNA using the following primers: F tion generated from the corresponding calibration curve, 5′-GGGGTACCAAGCTTTGCTGAATTGTCAAATTC which was made using the corresponding standard sub- CGTGG-3′ with Kpn Iand Hind III restriction sites, stance. Individual tissue samples above were taken from and R 5′-CCGCTCGAGTCTAGAATAGTGAGATGC 3 to 5 leaves or fruits for each line in triplicate. TGCTGCCA-3′ with Xho Iand Xba I restriction sites. The PCR products were cloned into the pHANNI- ABA measurement BAL vector in the sense orientation and anti-sense orien- ABA was extracted from 100 mg of pericarp tissues of tation into the Hind III-Xba I polylinker and the Kpn fresh ripe fruits (Br + 7 days) using 1 mL of solution I I-Xho I polylinker, respectively. Then the intron-spliced (80% methanol, 19.95% H O and 0.05% acetic acid, v/v). hairpin construct of SlIPT4 specific fragment under the The supernatant was collected, dried under a stream of transcriptional control of constitutive CaMV35S promoter N and dissolved in 0.5 mL petroleum ether to remove and OCS terminator was subcloned as Spe I-Sac Ifrag- the pigments. Then the subnatant was collected, dried ment into pCAMBIA1301 binary vector, in which the under a stream of N and dissolved in 0.5 mL solution II hygromycin resistance gene has been replaced by the neo- (40% methanol, 59.94% H O and 0.06% acetic acid, v/v). mycin phosphotransferase II (nptII) gene. Transgenic The HPLC analysis was carried out with 10 μL solution plants were generated by Agrobacterium tumefaciens-me- II-dissolved ABA using ultraviolet/visible detector on diated transformation. The positive transgenic lines were ACQUITY UPLC (Waters, USA). Spectra were collected checked by histochemical β-glucuronidase (GUS) strain- at 254 nm, and ABA contents were calculated through ing and PCR, and the silencing efficiency of SlIPT4 gene the linear regression equation generated from the cali- were detected by qRT-PCR. bration curve, which was made using the standard sub- stance of ABA (Sigma, USA). Individual tissue samples Drought treatment were taken from 3 to 5 fruits for each line in triplicate. The 4-week-old wild-type tomato plants were stopped watering. When the leaves showed serious wilting, the Results leaves of drought stress plants and control plants supplied SlIPT4 expression is predominant in ovary, sustained water normally were collected, respectively. Subsequently, enhancement during fruit ripening, and regulated by the drought treatment plants were supplied water again, drought stress and the leaves of 2, 6, 12 and 24 h after watering were col- Knowing the expression patterns of a gene sometimes is lected during the recovery process. The treatments were helpful for knowledge about its physiological function, performed with three biological replicates. thus the levels of SlIPT4 expression in tomato were Zhang et al. BMC Plant Biology (2018) 18:107 Page 4 of 12 comprehensively examined using qRT-PCR. In about the normal level at once after recovering watering, on the 8-week-old tomato plants, SlIPT4 mRNA was detectable contrary, it decreased more seriously. After 6 h of res- in all organs, and shown strong abundance in roots, toration, the SlIPT4 transcripts increased gradually stems and leaves, moderate accumulation in flowers and and reached 1.6 fold of that in the control plants at weak level in fruits (Fig. 1a). Given the confirmed roles 24 h. The results suggest that SlIPT4 may be involved of CKs in the process of leaf senescence and at the early in plant response to drought stress, where the phyto- stages of fruit development, SlIPT4 transcripts were ex- hormone ABA is the best known trigger of drought amined throughout leaf and fruit ontogeny. The mRNA tolerance in plants . accumulation of SlIPT4 is high in young leaves, and dis- tinctly and continuously downregulated along with the SlIPT4 knockdown in tomato accelerated leaf senescence process of leaf development and maturation (Fig. 1b). In To characterize the physiological function of SlIPT4 in flower, SlIPT4 expression is predominant in ovary, where tomato, a loss-of-function approach was implemented SlIPT4 mRNA keeps enormous accumulation at bud (− using RNAi strategy. A total of more than ten transgenic 2 dpa) and anthesis (0 dpa) stages, and displays dramatic lines were generated via Agrobacterium tumefaciens-me- downregulation from anthesis to post-anthesis (4 dpa) diated transformation. The most readily visible pheno- transition when fruit set is expected to occur (Fig. 1c). type was related to leaf senescence. The wild-type Subsequently, SlIPT4 expression maintains a moderate tomato leaves were still green and alive at later growth level at the mature green stage of fruit development. stage of about 8-week-old plants (Fig. 2a). By contrast, Interestingly, SlIPT4 expression displays a sustained en- the leaves of SlIPT4-RNAi transgenic lines turned into hancement along with fruit ripening, especially, obvious yellow color at expanded mature stage and displayed an sharp upregulation from orange stage to red stage when accelerated senescence phenotype (Fig. 2b), and most of lycopene biosynthesis is high active (Fig. 1d), which pro- leaves withered and abscised from plants at 8-week-old vides an important clue about its potential roles in the stage (Fig. 2a). The level of SlIPT4 transcript was signifi- process of tomato fruit ripening. cantly reduced by more than 70% in transgenic lines com- The expression of SlIPT4 was significantly decreased by pared with that in wild-type control plants (Fig. 2c), further 40% under drought stress compared to the control plants supporting that downregulation of SlIPT4 accounted for (Fig. 1e). Interestingly, SlIPT4 mRNA didn’treturn toward the phenotypes displayed in the transgenic lines. The ab c 1.8 80 1.2 Bud Anthesis Post-anthesis 1.2 40 0.8 0.6 0.4 0 0 Ro St Le Fl Fr Yl El Edl Ovary Sepal Stamen Petal 1.5 0.5 CK DT 2 h 6 h 12 h 24 h EMG MG Br Or Ri after watering Fig. 1 Expression patterns of SlIPT4 in tomato. Relative expression analysis of SlIPT4 was performed in different tissues (a), in leaves at different developmental stages (b), in different parts of flower at different developmental stages (c), in fruits at different stages (d), and in response to drought stress treatment (e) by qRT-PCR. Petals have been shed at the post-anthesis stage, so no data was shown at this stage. Data are expressed as relative values, based on the values of leaf (a), expending leaf (b), early mature green fruit (c and d), and corresponding control groups (e) taken as reference sample set to 1. Each value represents mean ± SE (standard error) of three replicates. Ro, root; St, stem; Le, leaf; Fl, flower; Fr, fruit. Yl, young leaf;El, expending leaf; Edl, expended leaf. EMG, early mature green; MG, mature green; Br, break; Or, orange; Ri, ripening. CK, control; DT, drought treatment; 2, 6, 12 and 24 h represent the hours after watering Relative mRNA (Fold) Relative mRNA (Fold) Relative mRNA (Fold) Relative mRNA (Fold) Relative mRNA (Fold) Zhang et al. BMC Plant Biology (2018) 18:107 Page 5 of 12 ab 1.2 SlIPT4 WT 0.8 0.4 ** ** ** RNAi RNAi WT WT RNAi-3 RNAi-8 RNAi-13 5 8 SAG12 WRKY53 ** ** ** ** ** 60 ** 2 30 ** ** ** WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 1.2 15 1.2 ** GLK2 Rbcs3A SGR 0.8 0.8 10 ** ** 5 0.4 0.4 ** ** ** ** ** ** WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 Fig. 2 SlIPT4 knockdown in tomato induced leaf senescence. a and b The phenotype of accelerated leaf senescence in SlIPT4-RNAi lines. Most leaves withered and abscised at 8-week-old plants (a), and leaves at expanded mature stage became yellow color (b). c SlIPT4 silencing efficiency was confirmed by qRT-PCR. d and e Relative expression levels of senescence marker genes, SAG12 and WRKY53, were tested by qRT-PCR. f Obvious reduction of total chlorophyll in expanded leaves of SlIPT4-RNAi lines as shown in b. Biological replicates were performed in triplicate, and the data are shown as mean ± SE. g and h and i Relative expression levels of GLK2, SGR, and Rbcs3A (regulating chlorophyll biosynthesis, chlorophyll degradation, and photosynthetic rate, respectively) were tested by qRT-PCR. For qRT-PCR, data are expressed as relative values based on the values of the control wild-type (WT) taken as reference sample set to 1. Each value represents mean ± SE of three replicates. Asterisks represent significant differences between SlIPT4-RNAi line and wild-type control (*P < 0.05, **P < 0.01, Student’s t test) senescence specificity of senescence-associated gene SAG12, transcription factor Golden2-like (GLK2) regulates posi- encoding a cysteine protease, makes this gene as a molecular tively chlorophyll biosynthesis through affecting chloro- marker to study the senescence process . The transcrip- plast development . STAY-GREEN (SGR) plays a tion factor WRKY53 regulates senescence specific gene ex- decisive impact on chlorophyll degradation in plants pression . Further the remarkable enhanced expression . Agreeing with the accelerated-senescence pheno- of SAG12 (GenBank no. XM_004233006) and WRKY53 type and the reduced chlorophyll accumulation in (GenBank no. XM_004244582), indicated the accelerated SlIPT4-RNAi leaves, the transcripts of GLK2 were re- senescence occurred in SlIPT4-RNAi leaves (Fig. 2d and e). markably decreased (Fig. 2g), while SGR expression was Knowing chlorophyll is predominant pigments in significantly upregulated (Fig. 2h), suggesting that SlIPT4 green leaves, plays an essential role in photosynthesis, silencing could disturb chlorophyll accumulation and is degraded during the process of leaf senescence, it through blocking chlorophyll biosynthesis and promot- prompted us to measure chlorophyll content in leaves. ing the degradation of chlorophyll in leaves. The de- At the expanded mature stage as shown in Fig. 2b, crease of photosynthetic rate is also an important SlIPT4 silencing resulted in an about 75% reduction of characteristic of leaf senescence. The dramatic downreg- total chlorophyll content in yellow leaves of SlIPT4-R- ulated expression of a key gene, ribulose-1,5-bispho- NAi lines compared with that in green leaves of sphate carboxylase small subunit (Rbcs3A) (Fig. 2i), in wild-type control plants (Fig. 2f). Considering chloro- regulating photosynthetic rate , indicated a reduced phyll homeostasis is mainly maintained through the dy- photosynthetic rate in SlIPT4-RNAi leaves. It is well namic balance between biosynthesis and degradation in known that CKs can inhibit leaf senescence , how- plants, the expression levels of two crucial genes regulat- ever, its exact regulation mechanism is still unknown. ing these processes were determined by qRT-PCR. The Here phenotype and molecular analyses of SlIPT4-RNAi Relative mRNA (Fold) Relative mRNA (Fold) Relative mRNA (Fold) Relative mRNA (Fold) Relative mRNA (Fold) Relative mRNA (Fold) Zhang et al. BMC Plant Biology (2018) 18:107 Page 6 of 12 transgenic tomatoes support the hypothesis that SlIPT4 carotenoids were measured in both SlIPT4-RNAi and is involved in this regulatory network of leaf senescence. wild-type fruits. Compared with wild-type control fruits, the contents of all carotenoids measured were reduced in SlIPT4 silencing in tomato caused orange fruits and SlIPT4-RNAi fruits (Fig. 3c-f). Total carotenoid levels were decreased lycopene accumulation reduced by about 35% when quantified using spectro- Although the role of CKs in regulating cell division at the photometric methods (Fig. 3c). However, HPLC-mediated early stages of fruit development is well known, and quantification of lycopene accumulation showed a decrease SlIPT4 gene exhibits very strong expression level in ovar- of more than 75% (Fig. 3d,Additionalfile 2:Table S2). Due ies and obvious dynamic expression in flowers during lycopene is the predominant pigment and endows the ripe bud-to-anthesis and anthesis-to-post-anthesis transitions fruit with deep-red color, we concluded that the enormous (Fig. 1c), the behaviors of fruit set and developmental reduction of lycopene accounted for the orange color process in all SlIPT4-silenced lines were indistinguishable in transgenic fruits. Meanwhile two derivatives of from the control plants. However, surprisingly, the fruits lycopene, β-carotene and lutein, were measured to as- of SlIPT4-RNAi lines couldn’t normally ripen and dis- sess the activity of lycopene catabolism. Similar to played orange surface at about 7 days after breaker stage, lycopene in SlIPT4-RNAi fruits, the contents of both while the fruits of wild-type control plants could become β-carotene and lutein were also significantly reduced typical deep-red at the same growth stage (Fig. 3a). To re- (Fig. 3e-f,Additional file 2: Table S2), suggesting the low veal whether SlIPT4 downregulation delayed the progress content of lycopene in SlIPT4-RNAi fruits was not be- of tomato fruit ripening, the orange fruits were held on cause of its biological catabolism. Therefore, repression of the plants. However, the orange fruits of SlIPT4-RNAi SlIPT4 expression leaded to the orange fruit phenotype lines didn’t switch to red color even when plants died (see through inhibiting lycopene biosynthesis. 0 day fruits in Fig. 3b). Moreover, these orange fruits also couldn’t change the color after a long-time storage of Expression of carotenoid biosynthetic genes were altered about 30 days at room temperature (Fig. 3b). These results in SlIPT4-silenced fruits stated clearly that SlIPT4 is involved in regulating color The carotenoid biosynthesis is mainly regulated at formation during tomato fruit ripening, and its downregu- gene transcriptional level during fruit ripening . lation caused never red fruits. Accordingly, to detect the mRNA levels of genes in- Given the dramatic increase of carotenoids, especially volved in lycopene biosynthesis and metabolism path- lycopene during fruit ripening in tomato, the related way is helpful to uncover the molecular regulation ab c Total carotenoids 0 Day ** ** ** 30 Day WT RNAi-3 RNAi-8 RNAi-13 WT RNAi WT RNAi d e f Lycopene Lutein ** ** ** ** ** 20 ** ** ** ** WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 Fig. 3 SlIPT4 downregulation in tomato resulted in orange fruits and decreased lycopene accumulation. a and b Phenotypes of fruits at about 7 days after break stage (a), and after holding on plants until plant died (0 day) and a storage of 30 days at room temperature (b). c, d, e and f A moderate reduction of total carotenoids (c) measured using spectrophotometer, and dramatic decrease of lycopene (d), β-carotene (e), and lutein (f) quantified via HPLC in SlIPT4-RNAi orange fruits. FW = fresh weight. The biological replicates (3–5 fruits per sample) were performed in triplicate, and the data are shown as mean ± SE. Asterisks represent significant differences between SlIPT4-RNAi line and wild-type control (*P <0.05, **P <0.01, Student’s t test) -1 µg g FW -1 µg g FW -1 µg g FW -1 µg g FW Zhang et al. BMC Plant Biology (2018) 18:107 Page 7 of 12 mechanism of orange color fruits caused by SlIPT4 PDS,and CrtISO genes was not the reasons for causing RNAi. As description above, the steps in lycopene biosyn- orange fruits in SlIPT4-RNAi lines, while the downregula- thesis pathway are orderly catalyzed by several crucial en- tion of ZISO and ZDS, especially the dramatic decrease of zymes encoded by PSY1, PDS, ZISO, ZDS,and CrtISO ZISO mRNAs, blocked the carotenoid biosynthesis path- genes, respectively (the pathway was shown in Fig. 6). way and resulted in the decreased accumulation of Here the transcript levels of these genes were tested in lycopene. 5-day fruits after break stage by qRT-PCR (Fig. 4). The In the process of lycopene metabolism, two key cyclases, first two genes, PSY1 and PDS, being responsible for col- lycopene β-cyclase (LCYB) and lycopene ε-cyclase (LCYE), orless phytoene synthesis and desaturation to generate catalyze the formation of β-ring and ε-ring, respectively 9,15,9′-cis-ζ-carotene, were markedly upregulated in [13, 14]. Although two products of lycopene metabolism, SlIPT4-RNAi fruits. The ZISO gene required for the isom- β-carotene and lutein, were reduced as described above erization of 9,15,9′-cis-ζ-carotene to 9,9′-cis-ζ-carotene, (Fig. 3e-f), the expression levels of LCYB and LCYE were was significantly downregulated by more than 80% in moderately increased in SlIPT4-RNAi lines (Fig. 4). This SlIPT4-RNAi fruits. The mRNA of ZDS, encoding a desa- paradox might due the enhanced catabolism of β-carotene turase that catalyzes 9,9′-cis-ζ-carotene to produce proly- and lutein, or that low lycopene accumulation caused the copene, was mildly reduced in SlIPT4-RNAi lines. reduction of lycopene metabolism products, which regu- Moreover, SlIPT4 overexpression under the control of lated the expression of their related catalyzing enzyme constitutive CaMV35S promoter increased the transcript genes through a negative feedback mechanism. accumulation of both ZISO and ZDS, and leaded to a little deep-redder fruits than wild-type control tomatoes (data SlIPT4 silencing caused a reduction of ABA content and not shown). The CrtISO and two CrtISO-like genes (CrtI- upregulated expression levels of ABA biosynthesis-related SO-L1 and CrtISO-L2) being responsible for the step of genes prolycopene to all-trans-lycopene in tomato, were upregu- Considering that the biosynthesis of ABA from the lated by different degrees in SlIPT4-RNAi lines. In conclu- oxidative cleavage of β-carotene is the main pathway sion, we thought that the upregulated expression of PSY1, in higher plants, and ABA feedback-regulates the 12 9 1.2 PSY1 PDS ZISO ** ** ** ** ** 8 6 0.8 ** 4 3 0.4 ** ** ** 0 0 0 WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 1.2 2 8 ZDS CrtISO CrtISO-L1 ** 1.5 6 0.8 ** 1 4 ** 0.4 0.5 2 0 0 0 WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 LCYE 3 2.5 3 CrtISO-L2 LCYB ** ** ** ** 2 ** 2 ** ** ** ** 1.5 1 1 0.5 0 0 0 WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 Fig. 4 Altered expression of genes in lycopene biosynthesis and metabolism pathway in SlIPT4-RNAi orange fruits. The relative level of transcripts were detected via qRT-PCR between wild-type and SlIPT4 RNAi lines. The data of wild-type (WT) were taken as reference and normalized to 1. Each value represents mean ± SE of three replicates. Asterisks represent significant differences between SlIPT4-RNAi line and wild-type control (*P < 0.05, **P < 0.01, Student’s t test) Relative mRNA (Fold) Relative mRNA (Fold) Relative mRNA (Fold) Zhang et al. BMC Plant Biology (2018) 18:107 Page 8 of 12 carotenoid biosynthesis [3, 42], the ABA content and the grain-filling period, thereby increasing crop yield. In expression levels of ABA biosynthesis-related genes were plants, it has been confirmed that phytohormones are tested to assess whether the decrease of β-carotene con- crucial in the regulation of leaf senescence, and among tent affected ABA biosynthesis in SlIPT4-RNAi lines. The the various plant hormones, CKs have received the max- obvious reduction of ABA content in SlIPT4-RNAi fruits imum attention for their roles in depressing leaf senes- (Fig. 5a), proved that the reason of the low content of cence . For instance, exogenous application of CKs lycopene metabolism products is not their catabolism to has the ability to delay the senescence of detached leaf produce ABA, but their weak biosynthetic activity caused in dark condition . Although CKs play key roles in by the very low accumulation of lycopene. In ABA biosyn- regulating leaf senescence, little is known about their thesis pathway, zeaxanthin epoxidase (ZEP) and 9-cis-e- exact regulation mechanism. Furthermore, endogenous poxycarotenoid dioxygenase (NCED) play key roles in CKs include several different forms, such as trans-zeatin higher plants. The ZEP catalyzes the formation of violax- (tZ), isopentenyladenosine (iPR), dihydrozeatin riboside anthin from zeaxanthin , and NCED controls the first (DZR), and isopentenyladenine (iP), each of them may committed and rate-limiting step of ABA biosynthesis play different roles in plants. It is puzzled which one or from carotenoid pathway . The upregulation expres- several CKs control the process of leaf senescence. The sion of both NEP and NCED (NCED1 and NCED2)inor- isopentenyltransferases (IPTs) are the rate-limiting en- ange fruits of transgenic lines (Fig. 5b-d), suggested that zymes catalyzing the initial step of CK biosynthesis, and ABA biosynthesis genes were negative feedback regulated are thought to have a different role in producing differ- by the level of ABA accumulation. ent CK among them . Thus it is very necessary to il- luminate the functions of IPTs. In the present study, we Discussion demonstrated that repressing expression of SlIPT4 accel- Leaf senescence is an internally programed phase with erated leaf senescence in tomato, which is active and the redistribution of micro- and macro-nutrients from useful to discover the molecular mechanism of CKs leaves to reproductive organs [40, 45–47]. Premature regulating leaf senescence. senescence in leaves generally results in reduced crop The prominent visible color change (from green to production and poor grain quality, while delaying leaf yellow) of leaf senescence is mainly caused by the deg- senescence can prolong the photosynthesis time and radation of green pigment chlorophyll . Chlorophyll provide sufficient assimilated carbon to grain during the catabolism is a multistep pathway regulated by many 2.5 ZEP 2 ** ** 1.5 * ** 0.5 WT RNAi-3 RNAi-8 RNAi-13 WT RNAi-3 RNAi-8 RNAi-13 NCED1 ** NCED2 ** ** ** ** ** WT RNAi-3 RNAi-8 RNAi-13 Fig. 5 Reduced accumulation of ABA in SlIPT4-RNAi orange fruits. a ABA content was measured using HPLC analysis. b, c and d Relative expression levels of ABA biosynthesis-related genes were tested by qRT-PCR. The data of wild-type (WT) were taken as reference and normalized to 1. Each value shown as mean ± SE of three replicates. Asterisks represent significant differences between SlIPT4-RNAi line and wild-type control (*P < 0.05, **P < 0.01, Student’s t test) -1 ABA content (µg g FW) Relative mRNA (Fold) Relative mRNA (Fold) Relative mRNA (Fold) Zhang et al. BMC Plant Biology (2018) 18:107 Page 9 of 12 factors . And among them, the SGR protein, charac- chloroplast and the cytosolic area surrounding chloro- terized from stay green mutant, plays crucial roles in the plasts uncovered by Žižková et al. , strongly supports regulation of chlorophyll degradation through dismant- our results that SlIPT4 is involved in regulating leaf sen- ling photosynthetic chlorophyll-protein complexes and escence and fruit color formation. thus allowing chlorophyll-breakdown enzymes to access Among lycopene biosynthesis-related genes, SlIPT4 their substrate [51–55]. Repression of SGR expression RNAi resulted in remarkable decrease of ZISO mRNA, delayed chlorophyll degradation in tomato fruits and moderate downregulation of ZDS transcript, and obvi- leaves, while constitutive expression of SGR accelerated ous upregulated expression of PSY1, PDS, and CrtISOs chlorophyll breakdown in leaves . In SlIPT4-silenced (Fig. 4). ZISO is first defined by the maize y9 and present leaves, the level of SGR mRNA was remarkably in- in single copy in tomato, Arabidopsis and grape [10, 56, creased by about 8–12 fold (Fig. 2h), consistent with the 57]. In tomato, ZISO widely expresses in flower, root, decreased content of total chlorophyll (Fig. 2f). Besides leaf and fruit, and maintains high expression level during enhanced degradation of chlorophyll, the chlorophyll fruit ripening . Silencing the expression of ZISO via biosynthesis-related gene, GLK2, was decreased in virus-induced gene silencing (VIGS) in tomato resulted SlIPT4-silenced leaves (Fig. 2g). Moreover, the decline in in pale-red fruits with a distinct reduction of lycopene photosynthetic capability is also the vital feature of leaf and a compensatory increased accumulation of phy- senescence, and the expression of Rbcs3A, encoding a toene, phytofluene, and ζ-carotene . Similarly, the re- positive regulator of photosynthesis, was significantly cessive mutant zeta (z ), a mutation in ZISO gene, downregulated (Fig. 3i), suggesting the reduced photo- blocks carotenoid biosynthesis in fruits at ζ-carotene with synthesis efficiency in SlIPT4-RNAi lines. Taken to- almost undetectable lycopene . Moreover, SlIPT4 gether, these data indicated that SlIPT4 is assuredly overexpression could enhance the mRNA levels of both necessary for controlling leaf senescence. ZISO and ZDS, and give rise to a little deep-redder fruits It was expected that repression of SlIPT4 expression (data not shown). Taken together, these data strongly sup- accelerated leaf senescence. However, it was out of our ported that downregulation of SlIPT4 expression in to- expectation that SlIPT4 knockdown did not cause any mato fruit inhibited carotenoids biosynthesis, especially obvious phenotypic changes during fruit developmental caused a sharp reduction of lycopene content, through stages, especially the cell division stage. Although the repressing the expression of ZISO. roles of CKs in fruit development are well known, not The puzzling complex regulatory loops regulating the all types of CKs have effects on this process. For ex- abundance of key transcripts in response to the oper- ample, after applications of exogenous CKs in unpolli- ation of the carotenoid biosynthesis pathway is existent nated ovaries of tomato, tZ, 6-benzylaminopurine (BA), in tomato, it was discussed previously by Kachanovsky and kinetin didn’t have any visible effect on fruit set and et al.  and Fantini et al. . The level of product growth, it was similar to untreated control ovaries with feedback regulates the transcript induction of its up- phenotype of neither abscission nor growth, however, stream genes in carotenoid biosynthesis pathway. For ex- application of N-(2-chloro-pyridin-4yl)-N′-phenylurea ample, the PDS promoter is induced when carotenoid (CPPU) induced growth . Thus, we think no visible accumulation is repressed in tomato leaves ; Prolyco- effect on fruit development in SlIPT4-RNAi lines may be pene or its metabolite was hypothesized as the signal due that there is potential functional redundancy among mediating PSY1 induction ; Fantini et al. thought a IPT genes during fruit development, or this kind of CK minimum level of prolycopene is required to attain PSY1 produced by SlIPT4 may not be pivotal for this process. transcript induction . The transcript level of ZISO CKs are thought to be important for fruit develop- was highly correlated with mRNAs of PDS, encoding the ment, especially cell division , to our knowledge, enzyme that produces the substrate for ZISO . The their roles in fruit ripening have no reports so far. Inter- ZISO and CrtISO were induced in PDS- and ZDS-si- estingly, the mRNA level of SlIPT4 gradually increases lenced fruits, respectively . The downregulation of during fruit ripening in tomato (Fig. 1d). More surpris- ZISO and ZDS, and the upregulation of other genes ingly, repressing the expression of SlIPT4 resulted in (PSY1, PDS, CrtISO, CrtISO-L1, and CrtISO-L2) involved producing orange fruits, which were never red (Fig. 3a-b). in carotenoid biosynthesis in SlIPT4-silenced fruits with As the predominant pigment in ripe tomato fruits, lyco- very low content of lycopene (Figs. 3 and 4), supports pene content was tremendously decreased (Fig. 3d, Add- the hypothesis of the existence of regulatory loops in the itional file 2: Table S2), suggesting SlIPT4 silencing carotenoid biosynthesis pathway. dramatically effected lycopene biosynthesis. During fruit It has been confirmed that ABA is mainly derived from ripening, chloroplasts convert to chromoplasts, which carotenoids pathway in higher plants, and low content of are pigment-filled plastids responsible for the bright ABA caused by disruption of carotenoid biosynthesis or colors. Thus the subcellular localization of SlIPT4 in the active biosynthesis of ABA induces PSY transcription Zhang et al. BMC Plant Biology (2018) 18:107 Page 10 of 12 through feedback regulation mechanism (reviewed in ref. SlIPT4 silencing and the sharp repression of carotenoid [3, 58]). For example, under abiotic stress of saline or biosynthesis might result in a reduced accumulation of drought, the increased demand for ABA to trigger stress ABA in SlIPT4-RNAi fruits. tolerance drives PSY expression to enhance carotenoid Consequently, based on the results and analysis above, biosynthesis pathway for providing more xanthophylls of we raised a proposed regulation model for the roles of ABA precursors in roots [60, 61]. In SlIPT4-RNAi fruits, SlIPT4 in the control of lycopene biosynthesis and leaf sen- the ABA accumulation was reduced due to the repression escence in tomato (Fig. 6). In this model, SlIPT4 gene regu- of carotenoid biosynthesis pathway (Fig. 5a), while the up- lates the lycopene biosynthesis via controlling the regulated expression of ABA biosynthesis-related genes expression level of ZISO. The ABA accumulation is regu- (ZEP, NCED1 and NECD2)(Fig. 5b-d) indicated that the lated by SlIPT4 through two potential ways: one way is that transgenic plants might try to restore the normal level of the alteration of carotenoid biosynthesis affects directly the ABA. Accordingly, the low accumulation of ABA in supply of ABA precursors; and another way is that ABA SlIPT4-RNAi fruits was likely to feedback regulate ca- accumulation is regulated by the potential correlation be- rotenoid biosynthesis pathway for trying to promote tween CKs and ABA. Then the alteration of ABA content the supply of ABA precursors through inducing PSY1 ex- feedback regulates the expression level of the first gene pression, which accords with this feedback regulation sys- PSY1 in carotenoid biosynthesis pathway. Though ABA tem between ABA content and PSY1 expression. was discovered to be involved in leaf senescence, however, The decreased content of CKs in ipt1 3 5 7 mutants or due to the known role of CKs in leaf senescence, we overexpression transgenic plants of CKX, encoding a thought that the SlIPT4-mediated leaf senescence might be cytokinin oxidase/dehydrogenase catalyzing the irrevers- mainly attributed to affect the corresponding CK biosyn- ible degradation of CKs, could significantly reduce ABA thesis in tomato. Nevertheless, the exact mechanism accumulation in Arabidopsis thaliana . SlIPT4 ex- through which the effects of SlIPT4 are exerted in tomato pression is very sensitive to drought stress (Fig. 1e), leaves and fruits needs and deserves further investigation. where ABA is the best known trigger of drought toler- ance in plants . These results suggest that SlIPT4 Conclusion may be associated with ABA biosynthesis. Accordingly, Most studies devoted so far to IPT genes focused on both the potential reduction of CK content caused by their roles in the known cytokinin-related process, such GGPP PSY1 ABA 15-cis-Phytoene PDS NCED ZISO Violaxanthin ZDS SlIPT4 Prolycopene ZEP CrtISO All-trans-lycopene Leaf senescence LCYB Zeaxanthin Pathways of biosynthesis Several enzymatic step Positive regulation Negative regulation Fig. 6 Proposed model for SlIPT4 regulating lycopene biosynthesis and leaf senescence in tomato. SlIPT4 regulates the carotenoid biosynthesis via controlling the expression level of ZISO, and modulates leaf senescence through affecting the corresponding CK biosynthesis. The alteration of carotenoid biosynthesis affects directly the supply of ABA precursors, and also ABA accumulation is regulated by the correlation between CKs and ABA. ABA feedback regulates the expression level of the first gene PSY1 in carotenoid biosynthesis pathway Zhang et al. BMC Plant Biology (2018) 18:107 Page 11 of 12 as cell division, apical dominance, leaf senescence and Author details Bioengineering College, Chongqing University, Chongqing 400044, China. fruit development, as well as in response to biotic and School of Life Sciences, Chongqing University, Chongqing 400044, China. abiotic stress. Here, we uncovered the functional roles of SlIPT4 in not only leaf senescence but also the regula- Received: 7 December 2017 Accepted: 24 May 2018 tion of lycopene biosynthesis in tomato. 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