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The involvement of cytokinin and nitrogen metabolism in delayed flag leaf senescence in a wheat stay-green mutant, tasg1
Plant Science 278 (2019) 70–79
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Research Article
The involvement of cytokinin and nitrogen metabolism in delayed flag leaf senescence in a wheat stay-green mutant, tasg1
T
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Wenqiang Wanga,b, ,1, Qunqun Haoc,d,1, Wenlong Wangb,1, Qinxue Lib, Fengjuan Chend, Fei Nid, ⁎ ⁎ Yong Wangb, Daolin Fuc, Jiajie Wud, , Wei Wangb, a
College of Life Sciences, Zaozhuang University, Zaozhuang, 277000, China State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, 271018, Shandong, China c Department of Plant Sciences, University of Idaho, Moscow, ID, 83844, USA d State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai’an, 271018, Shandong, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Leaf senescence CK metabolism N metabolism RNA-seq Stay-green Wheat
In the present study on a wheat stay-green mutant, tasg1, we found that its delayed senescence at the late filling stage was related to the high cytokinin (CK) and N contents. RNA sequencing suggested that several genes may be responsible for the different senescence processes between wild-type (WT) and tasg1 plants. WT and tasg1 seedlings were treated with NH4NO3, lovastatin, and 6-benzylaminopurine (BAP), and the results suggested that the feedback of CK with N content regulated the leaf senescence in the tasg1 plants. Furthermore, a knock-out of the candidate gene cisZOGT1 (catalytic O-glucosylation in cis-zeatin) in the wheat mutant pool ‘Kronos’ exhibited delayed senescence at the late grain filling stage. Overall, our results suggested the cisZOGT1 gene has an important role in regulating wheat leaf senescence by regulating CK and N metabolism. At the same time, CK and N metabolism involved in delayed flag leaf senescence of tasg1 may be by a feedback pattern.
1. Introduction Leaf senescence is generally the last stage of leaf development, during which a fully-expanded leaf progresses to its death. Plant senescence is an internally programmed degenerative process, and premature leaf senescence can result in a low grain yield [1]. Compared to WT plants, stay-green or non-yellowing mutants of various plant species have been reported to maintain the greenness of their leaves during senescence for a longer time, and are ideal materials for studying the mechanisms underlying plant senescence [2], which, to date, remains unclear. Cytokinins (CKs), which are widespread in nature, can be distinguished as isopentenyladenine (iP)-, trans-zeatin (tZ)-, cis-zeatin (cZ)- or dihydrozeatin-type derivatives. The rate-limiting step of isoprenoid CK biosynthesis is catalyzed by isopentenyltransferases (IPTs) [3]. The initiation of senescence involves alterations in the levels of CK [4]. Exogenous application of 6-benzylaminopurine (6-BA, a synthetic CK), at concentrations ranging from 2 to 200 μM, was reported to almost completely prevent petunia corolla senescence [5]. Transgenic expression of the IPT gene in maize, which encodes an isopentenyl
transferase, increased the CK levels and resulted in delayed senescence [6]. Moreover, senescence-regulated expression of IPT in rice was observed to delay the senescence of leaves, with regard to both the chlorophyll degradation and photosynthetic capacity [7,8]. The genetic control of N remobilization is linked to the regulation of leaf senescence [9]. Stay-green, the ability to retain green areas in the grain during filling, has been found to be related to the supply–demand balance of N during grain filling in Sorghum [10,11]. Modifying the N remobilization efficiency to delay senescence in feed wheat cultivars could favor a longer stage of active photosynthesis during grain filling, and might result in a higher grain yield [12]. Cultivars with prolonged N uptake under N-deficient conditions characteristically have delayed senescence in older leaves [13]. The side-chain modifying steps of CK, such as O-glycosylation, are expected to have a direct bearing on CK-mediated processes [14]. Veach et al. [15] reported that cisZOGT1 catalyzed O-glucosylation in the N6-sidechain of cis-zeatin. Compared to the highly active transzeatin, cis-zeatin is generally regarded as a CK with little or no activity [16]. Rodó et al. [17] also put forward that the characteristics of maize overexpressing the cisZOGT1 gene resembled those of CK-deficient
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Corresponding authors. Corresponding author at: College of Life Sciences, Zaozhuang University, Zaozhuang, 277000, China. E-mail addresses: [email protected] (W. Wang), [email protected] (J. Wu), [email protected] (W. Wang). 1 These authors contributed equally to this work. ⁎⁎
https://doi.org/10.1016/j.plantsci.2018.10.024 Received 30 August 2018; Received in revised form 18 October 2018; Accepted 31 October 2018 Available online 02 November 2018 0168-9452/ © 2018 Elsevier B.V. All rights reserved.
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measured according to the method of Lillo [21].
plants, whereas, a delayed senescence phenotype was exhibited in transgenic maize. Although some studies suggested possible roles for cis-zeatin, its physiological significance remains unclear. A wheat stay-green mutant, tasg1, was previously generated through mutagen breeding in our laboratory with the application of ethyl methane sulfonate (EMS) to the WT of the ‘HeSheng2′ cultivar. Our studies revealed that the stay-green phenotype of tasg1 was associated with altered CK metabolism [1]. Moreover, sucrose metabolism, regulated by CKs, was also involved in the delayed senescence of tasg1 [18]. In the present study, using RNA-seq, we found differentially expressed genes (DEGs) related to CK and N metabolism between WT and tasg1. Then, we used tasg1, 2885 (a durum wheat mutant) to determine two details of CK and N metabolism in the delayed senescence phenotype. 1) The feedback between CK and N metabolism in the delayed senescence of tasg1; 2) The regulation of wheat leaf senescence by cisZOGT1, and the involvement of CK and N metabolisms in cisZOGT1-regulated senescence.
2.6. Determination of free amino acid content The methods for the extraction and measurement of free amino acids were modified according to Li et al. [22]. The external standards for free amino acid were procured from Agilent Technologies (Waldbronn, Germany). 2.7. Quantitative reverse transcription PCR analysis Total RNA was extracted from the second leaf with TransZol (TransGen Biotech, China), and treated with DNase I (RNase-free, Promega). qRT-PCR was carried out in a 25 μL reaction volume containing 2× TransStart Top Green q-PCR SuperMix (TRANS, China). Quantitative analysis was performed using the Bio Rad CFX Manager system. This method normalizes the expression of a specific gene versus a control reference with the formula 2−△△CT. Actin and tubulin were evaluated as the control genes. Information on the genes analyzed is listed in Table S1.
2. Materials and methods 2.1. Field experiments
2.8. RNA sequencing and data analysis Field experiments were carried out at the farm of Shandong Agricultural University, China. The growth and development of the wheat plants were managed according to conventional agricultural techniques. Six interspersed plots (4 m2) were selected randomly in the field. Each plot was used for sample collection. The experiment was conducted at least in triplicate.
We collected two types of samples from the WT and tasg1 plants at the filling stage (28 d). Three biological replicates were used for each tissue genotype for the RNA sequencing. Total RNA was extracted from each sample using TRIzol reagent, following the manufacturer’s specifications (Invitrogen). The quality and quantity of each RNA sample was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). The mRNAs were isolated from the total RNA using Dynabeads mRNA DIRECT Kit (Invitrogen) and were fragmented into short fragments using a fragmentation buffer. Using these short fragments as templates, random primers, and a SuperScript double-stranded cDNA synthesis kit (Invitrogen), double-stranded cDNA was synthesized. Ligated fragments were then generated by a series of reactions that included purification of the PCR products, end repair, dA-tailing, and ligation of the Illumina adapters. After agarose gel electrophoresis, suitable fragments were selected for PCR amplification. The final library was evaluated by quantitative RT-PCR with a StepOne Plus RealTime PCR system (Applied Biosystems, Foster City, CA, USA). Sequencing reactions were performed on the Illumina HiSeq platform (PE 150) by the Berry Genomics Company (Beijing, China). Functional annotation and enrichment analysis of the differentially expressed genes (DEGs) was conducted using the Gene Ontology (GO) database (http://www.geneontology.org/). Information on the gene annotation is listed in Table S2.
2.2. Laboratory experiments WT and tasg1 seeds were germinated on filter paper, moistened with water for 24 h at 25 ± 1 °C after being sterilized with 0.2% sodium hypochlorite. Thereafter, the seeds were grown at 25 °C under a 16 h light/8 h dark illumination cycle (light intensity 300 μmol m−2 s-1) and 70% relative humidity. A nutrientless hydroponic system was used to induce leaf senescence in the wheat seedlings. The tasg1 plants showed a significantly delayed senescence phenotype in the late growth stage (11 d) compared to the WT plants. For BAP treatment, the wheat seedlings were sprayed by a 100 μM BAP solution (Sigma) with 0.02% (v/v) Tween-20. 2.3. Measurement of CKs The methods for the extraction and purification of CKs were modified according to Wang et al. [1]. The antigens, isopentenyl adenine (IPA), zeatin riboside (ZR), dihydrozeatin riboside (DHZR), and GA4 used in ELISA, and the mouse monoclonal antibodies against them were produced at the Phytohormones Research Institute of China Agricultural University.
2.9. Histochemical detection To visualize the dead cells, the detached leaves were stained with trypan blue (TB) [23]. The leaves were detached and submerged in 0.05% lactophenol–TB solution (0.05% TB, 25% [w/v] lactic acid, 25% water-saturated phenol, and 50% ethanol) at 70 °C for 10 min, and then heated in boiling water for 5 min and left overnight for staining. Destaining was subsequently performed in a 2.5 g μL−1 chloral hydrate solution for 3 d to reduce the background staining.
2.4. Measurement of the N content The leaf N content was determined according to the Kjeldahl method [19]. 2.5. Determination of NR, NiR, and GS activity
2.10. Chlorophyll content To determine the activity of different enzymes, leaves (0.1 g) were homogenized in 100 mM potassium phosphate buffer (pH 7.4), containing 7.5 mM cysteine, 1 mM EDTA, and 1.5% (w/v) casein using a chilled mortar and pestle. The homogenate was centrifuged at 20,000 × g for 15 min at 4 °C. The activities of Nitrate reductase (NR) and Nitrite reductase (NiR) were determined according to the method of Plett et al. [20]. The activity of Glutamine synthetase (GS) was
The chlorophyll contents were determined based on the method of Lichtenthaler [24]. 2.11. Statistical analysis All analyses were conducted, at least, in triplicate. The IBM SPSS 71
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Fig. 1. Different time-course dynamics during the filling stage between the wild-type (WT) and tasg1 plants. The parameters include the phenotype at different daypoints after flowering (A); the relative water content (B); the phenotype of flag leaves (28 d) (C); and the IPA (D), ZR (E), DHZR (F), and N (G) contents. Values are means ± SD based on thirty replicates. FW, fresh weight. DW, dry weight. Error bars indicate standard deviations.
plants (Fig. S1D). We also determined the expression of three senescence-regulated genes, namely, TaSAG1 (Fig. 2A), TaSAG2 (Fig. 2B), and TaSAG4 (Fig. 2C). Their expressions in the tasg1 plants were down-regulated compared to those in the WT plants. However, the expressions of CK biosynthesis-regulated genes, TaIPT2 (Fig. 2D), TaIPT3 (Fig. 2E), and TaIPT8 (Fig. 2F), were significantly higher in the tasg1 plants than in the WT plants. Furthermore, the expressions of N transport-regulated genes, TaNRT1.1 (Fig. 2G), TaNRT1.3 (Fig. 2H), and TaNRT1.4, (Fig. 2I) and amino acid synthesis-regulated genes, TaAlaAT (Fig. 2J), TaGS1 (Fig. 2K), and TaGS2 (Fig. 2L), were also significantly higher in the tasg1 plants than in the WT plants. These results suggest that both CK and N metabolism may be involved in the delayed senescence of tasg1, compared to that in its WT plant.
Statistics program was used to perform the statistical analyses. All comparisons were analyzed using factorial ANOVAs. Differences between the means among the wheat lines or treatments were compared using Duncan’s multiple range tests, at 0.05 probability levels. 3. Results 3.1. Dynamic delayed senescence phenotype of tasg1 plants at the filling stage We observed the dynamic phenotype throughout the filling stage of WT and tasg1 plants. We found that the senescence became evident at 21 d, after flowering in the WT plants (Fig. 1A). However, the senescence of tasg1 plants did not appear until day 24 of flowering (data not shown). Compared to the WT plants, the tasg1 plants had obviously delayed senescence at the late stages (28 d, 32 d, respectively) (Fig. 1A). Moreover, at the early stage (before 21 d), there was no significant difference in the relative water content between the WT and tasg1 plants, but at the late filling stage, it was 12.6% and 20.4% higher on days 28 and 32, respectively, in tasg1 than in WT (Fig. 1B). Based on the differences in the senescence phenotype between tasg1 and WT as shown in Fig. 1A, and the relative water content as shown in Fig. 1B, we selected the flag leaf at 28 d as the experimental material for the subsequent study (Fig. 1C).
3.3. RNA-seq analysis of the differential expression of genes related to CK and N metabolisms between WT and tasg1 plants To systematically analyze the contribution of CK and N metabolism to delayed leaf senescence in tasg1 plants, we performed RNA-seq using the flag leaves of WT and tasg1 plants, at 28 d after flowering. Compared to the WT samples, the transcripts in tasg1 samples had 8373 DEGs, with their expression differing by two-fold or more; of these, 3906 genes were down-regulated and 4467 genes were up-regulated (Fig. 3A and B, Table S2). To validate these DEGs, a qRT-PCR assay was performed for 19 randomly selected genes. The results shown in Fig. 3C showed the consistency between the RNA-seq and qRT-PCR data, indicating the credibility of the RNA-seq data. A total of 526 transcription factors (TFs) were differentially expressed, of which 238 were downregulated and 288 were up-regulated in the tasg1 plants compared to their expression levels in the WT plants (Fig. S2). The expression levels of the C2C2-YABBY family members were down-regulated, whereas those of the mTERF, Tify, and HSF family members were up-regulated. The expressions of some members in transcription factors families, such as MYB, bHLH, NAC, WRKY, and AP2/EREBP families, were either down-regulated or up-regulated. GO analysis was performed for the DEGs using the GO database,
3.2. Changes in CK and N metabolisms in tasg1 at the filling stage We measured the contents of CKs, including isopentenyl adenine (IPA), zeatin riboside (ZR), and dihydrozeatin riboside (DHZR), in tasg1 and WT at the filling stage. The contents of IPA, ZR, and DHZR were 53.8%, 17.6%, and 38.2% higher in tasg1 plants than in WT, respectively (Fig. 1D–F). Furthermore, the total N content in the tasg1 plants was about two-fold that in the WT plants (Fig. 1G). Simultaneously, the activities of NR, NiR, and GS showed a trend similar to that of N content; they were approximately 2–3-fold in the tasg1 plants compared to those in the WT plants (Fig. S1A–C). The free amino acid concentrations in the tasg1 plants were also significantly higher than those in the WT 72
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Fig. 2. Relative expression of genes related to senescence, CK biosynthesis, N transport, and amino acid synthesis, including TaSAG1 (A), TaSAG2 (B), TaSAG4 (C), TaIPT2 (D), TaIPT3 (E), TaIPT8 (F), TaNRT1.1 (G), TaNRT1.3 (H), TaNRT1.4 (I), TaAlaAT (J), TaGS1 (K), and TaGS2 (L) in the flag leaves (28 d) of wild-type (WT) and tasg1 plants at the filling stage. Values are means ± SD based on six replicates. Error bars indicate standard deviations.
Fig. 3. RNA-seq analysis of the transcriptome of flag leaves (28 d). The parameters include differentially expressed genes (DEGs) (A), changes in the gene expression profile of flag leaves (28 d) between wild-type (WT) and tasg1 plants (B), confirmation of RNA-seq by qRT-PCR data (C). Values are means ± SD based on three replicates. 73
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Fig. 4. Histograms of gene ontology annotation of CK metabolism in flag leaves (28 d) and wild-type (WT) and tasg1 plants at the filling stage. The parameters include the percentage of DEGs in biological process (A), biological process (B), the percentage of DEGs in molecular function (C), and molecular function (D). Values are mean ± SD based on three replicates.
the nitrogen fixation, ammonia assimilation cycle, cellular amino acid metabolic process, glutathione biosynthetic process, glutamine biosynthetic process, and glutamate metabolic process (Fig. 5A and B; Table S2). Moreover, for the molecular functions, among the downregulated genes, those encoding the cytokinin dehydrogenase and oxidoreductase were clearly over-represented in this category (Fig. 5C; Table S2). However, among the up-regulated genes, those encoding glutamate-ammonia ligase, ammonia ligase, acid-ammonia ligase, and glutathione synthase were clearly over-represented in the molecular function category (Fig. 5D; Table S2). Overall, the RNA-seq results shown in Figs. 3 and 5 also suggest that both CK and N metabolism may be involved in the delayed senescence of the tasg1 seen in Figs.1 and 2.
which provides information on the annotation of biological processes and molecular functions (http://www.geneontology.org/). Specifically, we analyzed the biological processes and molecular functions of CKs. For the biological process, obvious over-representation was observed for CKs metabolic processes, including cytokinin metabolic process, cellular hormone metabolic process, cytokinin catabolic process, and hormone metabolic process (Fig. 4A and B; Table S2). Moreover, for the molecular functions, among the down-regulated genes, a large number of genes encoding the cytokinin dehydrogenase and oxidoreductase were clearly over-represented in this category (Fig. 4C; Table S2). However, among the up-regulated genes, only a few genes encoding the cytokinin dehydrogenase and oxidoreductase were clearly over-represented in the molecular function category (Fig. 4D; Table S2) Furthermore, we also analyzed the biological processes and molecular functions of N metabolism. For the biological process, obvious over-representation was observed for N metabolic processes, including 74
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Fig. 5. Histograms of gene ontology annotation of N metabolism in flag leaves (28 d) of WT and tasg1 plants at the filling stage. The parameters include the percentage of DEGs in biological process (A), biological process (B), the percentage of DEGs in molecular function (C), and molecular function (D). Values are mean ± SD based on three replicates.
with NH4NO3, the activities of NR, NiR, and GS were significantly higher than those in the untreated plants (Fig. S3A–C). The expression levels of the nine genes related to CK biosynthesis, N transport, and amino acid synthesis were also determined. Under normal conditions, the expression levels of TaIPT2 (Fig. S4A), TaIPT3 (Fig. S4B), TaIPT8 (Fig. S4C), TaNRT1.1 (Fig. S4D), TaNRT1.3 (Fig. S4E), TaNRT1.4 (Fig. S4F), TaAlaAT (Fig. S4G), TaGS1 (Fig. S4H), and TaGS2 (Fig. S4I) were significantly higher in the tasg1 plants than in the WT plants. Furthermore, in the NH4NO3-treated WT and tasg1 plants, the expression levels of genes involved in N transport (TaNRT1.1, TaNRT1.3, and TaNRT1.4) and amino acid synthesis (TaAlaAT, TaGS1, and TaGS2), as well as those involved in CK biosynthesis (TaIPT2, TaIPT3, and TaIPT8) were significantly up-regulated in the WT and tasg1 plants compared to those in the untreated plants. These results suggest that CK metabolism was regulated by N content and/or N metabolism, which in turn affect wheat leaf senescence. To further substantiate the role of CKs in regulating leaf senescence, and to understand the involvement of CKs in N metabolism, the tasg1 plants were treated with 40 μM lovastatin (The seedlings were cultivated in a 40 μM lovastatin solution when they were grown to 7 days), which is a specific inhibitor of CK biosynthesis, and with 40 μM lovastatin along with 0.1 M NH4NO3. Under normal growth conditions, the tasg1 plants exhibited a delayed senescence phenotype compared to that of WT plants (Fig. 7A). The TB staining indicated that cell death
3.4. The feedback between CK and N content in the regulation of senescence in tasg1 plants To understand the involvement between the metabolisms of CK and N in the regulation of leaf senescence in tasg1, we externally controlled the N content and regulated the CK metabolism. To induce leaf senescence, we used two-week-old wheat seedlings in a nutrientless hydroponic system [1]. To control the N contents, WT and tasg1 plants were treated with 0.1 M NH4NO3. The seedlings were cultivated in a 0.1 M NH4NO3 solution when they were grown to 7 days. Since leaf senescence is usually accompanied by cell death, we examined the effect of NH4NO3 on cell death by Trypan Blue (TB) staining because the function of TB is to dye dead cells [25]. The WT and tasg1 plants exhibited a delayed senescence phenotype, and had reduced cell death (Fig. 6A and B). Moreover, the chlorophyll contents were also significantly increased in the WT and tasg1 plants when treated with NH4NO3 (Fig. 6C). We also measured the contents of the different CKs (IPA, ZR, and DHZR) and total N in the WT and tasg1 plants. The NH4NO3 treatment significantly increased the N content (Fig. 6G), as well as that of IPA (Fig. 6D), ZR (Fig. 6E), and DHZR (Fig. 6F) in the WT and tasg1 plants. We also examined the activity of NR, NiR, and GS, which are related to N metabolism (Fig. S3). Under normal conditions, the activities of NR, NiR, and GS in the tasg1 plants were significantly higher than those in the WT plants. However, when the WT and tasg1 plants were treated
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Fig. 6. Effect of NH4NO3 on wild-type (WT) and tasg1 seedling leaves at 11 d of growth. The parameters include the phenotype (A), trypan blue staining (B), chlorophyll content (C), isopentenyl adenine (IPA) content (D), ZR content (E), dihydrozeatin ribosideDHZR content (F), and N content (G). Values are means ± SD based on three replicates. FW, fresh weight. DW, dry weight. Error bars indicate standard deviations.
We further examined the expression of the genes related to CK biosynthesis, N transport, and amino acid synthesis (Fig. S5). Under normal conditions, the expression levels of TaIPT2 (Fig. S5 A), TaIPT3 (Fig. S5B), TaIPT8 (Fig. S5C), TaNRT1.1 (Fig. S5D), TaNRT1.3 (Fig. S5E), TaNRT1.4 (Fig. S5F), TaAlaAT (Fig. S5G), TaGS1 (Fig. S5H), and TaGS2 (Fig. S5I) were significantly higher in the tasg1 plants than in the WT plants. When treated with lovastatin, the expression levels of the genes involved in CK biosynthesis (TaIPT2, TaIPT3, and TaIPT8), N transport (TaNRT1.1, TaNRT1.3, and TaNRT1.4), and amino acid synthesis (TaAlaAT, TaGS1, and TaGS2) were significantly decreased in the tasg1 plants (Fig. S5). However, when treated with lovastatin plus NH4NO3, the expression of these genes was up-regulated (Fig. S5). We also examined the expression of the genes related to CK signaling (Fig. S6). However, when treated with lovastatin plus NH4NO3, the ARR6 (type-A response regulator) (Fig. S6A) was expressed at a lower level in tasg1 plants. In contrast, the expression of ARR18 (type-B response regulator) (Fig. S6B) was up-regulated in tasg1 plants. This result showed that application of N not only regulated CK biosynthesis pathway but also CK signaling pathway. Moreover, when the WT and
was obviously reduced in the tasg1 plants (Fig. 7B). When the tasg1 plants were treated with lovastatin, they exhibited a premature phenotype and increased cell death (Fig. 7A and B). However, application of NH4NO3 partially restored the premature senescence phenotype and reduced cell death of tasg1 with lovastatin treatment (Fig. 7A, B). The chlorophyll content corroborated with the phenotypic information (Fig. 7C). In addition, we measured the contents of the different CKs and total N levels. Under normal conditions, the IPA content (Fig. 7D), ZR (Fig. 7E), DHZR (Fig. 7F), and total N (Fig. 7G) in the tasg1 plants were approximately 1.06-, 1.83-, 1.86-, and 1.29-fold higher than those in the WT plants, respectively. However, when treated with lovastatin, their contents were significantly decreased (Fig. 7G) in the tasg1 plants. Furthermore, when treated with lovastatin plus NH4NO3, the contents of different CKs and total N were significantly increased in the tasg1 plants (Fig. 7D–G). We also examined the activities of NR, NiR, and GS in the WT and tasg1 plants (Fig. S3). When treated with lovastatin, their activities were significantly decreased in the tasg1 plants. However, when treated with lovastatin plus NH4NO3, their activities slightly increased in the tasg1 plants (Fig. S3D–F).
Fig. 7. Effect of lovastatin (lov, CKs inhibitor), and lovastatin plus NH4NO3 on wild-type (WT) and tasg1 seedling leaves at 11 d of growth. The parameters include the phenotype (A), trypan blue staining (B), chlorophyll content (C), isopentenyl adenine (IPA) content (D), ZR content (E), dihydrozeatin ribosideDHZR content (F), and N content (G). Values are means ± SD based on three replicates. FW, fresh weight. DW, dry weight. Error bars indicate standard deviations. 76
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this study, tasg1 plants exhibited a markedly delayed senescence phenotype at the late filling stage (Fig. 1A). N is an essential macronutrient for plant growth and development. The main symptom of N deficiency in plants is leaf senescence, which is a result of pigment loss, protein degradation, and lipid peroxidation [29]. Many studies have shown that N also plays an important role in the regulation of plant senescence [30,31]. In the present study, in addition to the contents of three types of CK, the N content in the flag leaves of tasg1 plants was also significantly higher than that in the WT plants at the late filling stage (Fig. 1C–G). Nitrate reductase, NiR, and GS are the three key enzymes involved in protein synthesis, and contribute to the growth of plants [20]. The activities of NR, NiR, and GS indicate that tasg1 plants have strong N assimilation and NH4+ transport capacities (Fig. S1). Studies have shown that senescence-associated genes (SAGs) are transcriptionally up-regulated during senescence [32]. The expression of TaSAGs further advanced the delayed senescence phenotype in the tasg1 plants (Fig. 2A–C). The expression of mRNAs related to CK biosynthesis, N transport, and amino acid synthesis indicates that both the CK and N metabolisms were changed in the flag leaves of tasg1 plants compared with those in the WT (Fig. 2D–L). RNA-seq was used to analyze the different expression of genes involved in CK and N metabolisms between tasg1 and its WT. Compared to WT plants, only a few genes encoding the cytokinin dehydrogenase and oxidoreductase were clearly over-represented in tasg1 plants in the molecular function category (Fig. 4C and D). The GO analysis of CK metabolism also supported the data of the increased CKs in tasg1 plants at the late filling stage, which can regulate the senescence of plants (Figs. 3, 4; Table S2). Notably, the GO analysis of N metabolism further revealed the difference in N metabolism between tasg1 and WT plants. Besides, a large number of genes encoding the N synthesis and transport in tasg1 at the late filling stage were examined (Figs. 3, 5; Table S2). From the results in Figs. 1–5, we suggest that CK and N metabolisms affect the delayed senescence of tasg1 plants. Transcriptional activators and repressors play crucial roles in mediating the signalling pathway of leaf senescence [25]. Previous studies have identified a few transcriptional regulators of H2O2-induced leaf senescence, including HSF, AP2/EREBP, and NAC [33,34]. Intriguingly, the expression levels of several TFs, including HSF and AP2/EREBP, were enriched in the up-regulated group (Fig. S2). These results imply that H2O2 scavenging might be involved in delayed leaf senescence in tasg1 plants. Our previous study also indicated that the H2O2 content in
tasg1 plants were treated with BAP, the WT and tasg1 plants exhibited a delayed senescence phenotype (Fig. S7A). At the same time, not only N content but also the expression levels of N metabolism genes (TaNRT1.1, TaNRT1.3, TaNRT1.4, TaAlaAT, TaGS1, and TaGS2) were significantly higher in WT and tasg1 plants (Fig. S7B-H). Taken together, these results suggested that CKs metabolism was also involved in N metabolism in regulating wheat leaf senescence. 3.5. The role of cisZOGT1 in delayed plant senescence We analyzed the DEGs for cytokinin metabolic pathways (about 8fold) between WT and tasg1. cisZOG1 (Traes_2 AL_21065D298) and CKX1 (Traes_3 AS_BEB2E9AA9) were screened in this study. The function of CK dehydrogenases1 (CKX1) gene has been widely reported in previous studies. CK degradation requires CKXs, which remove CK unsaturated isoprenyl side chains [26]. At the same time, overexpression of CKX1 leads to stronger CK deficient phenotype than that of other CKX genes [27]. Therefore, we focus on cisZOGT1 gene in this study. RNA-seq data showed that the expression of cisZOGT1 in tasg1 was significantly lower than that of WT (about 14%). This cisZOGT1 gene encoded a cis-zeatin O-glucosyltransferase 1. The results of qRTPCR further proved the lower expression of the cisZOGT1 gene in tasg1 than in WT (Fig. S8). Then, a knock-out of the cisZOGT1-B locus was screened from the durum wheat mutant pool ‘Kronos’ (Fig. S9). Therein, mutant 2885 lines, including 2885-1, 2885-2, and 2885-3 exhibited delayed leaf senescence (Fig. 8A). Moreover, the chlorophyll content in flag leaves of 2885 lines was significantly higher than that in WT at the late filling stage (Fig. 8B). The total N (Fig. 8C), ZR (Fig. 8E), and DHZR (Fig. 8F) contents in the mutant 2885 plants were also approximately 1.29-, 1.32-, and 1.11-fold higher than those in the WT, but no obvious differences were observed in IPA (Fig. 8D). 4. Discussion 4.1. CK and N metabolisms affect delayed leaf senescence in tasg1 plants Leaf senescence is a critical step in the life cycle of an annual plant. During the process, the apparent visual characteristics of leaf senescence are the loss of chlorophyll and eventual abscission [28]. In a previous report, we demonstrated that the altered CK metabolism was involved in the stay-green phenotype of the wheat mutant, tasg1 [1]. In
Fig. 8. The phenotype of WT and mutant 2885 during the late growth stage. The parameters include the phenotype (A), the chlorophyll content (B), N (C), isopentenyl adenine (IPA) (D), ZR (E), and dihydrozeatin ribosideDHZR (F). Values are means ± SD based on three replicates. FW, fresh weight. DW, dry weight. Error bars indicate standard deviations. 77
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DHZR contents in the mutant 2885 lines were higher than those in their respective WT plants (Fig. 8C, E, and F). These results reinforce the opinion that CK and N levels regulated by cisZOGT1 were important to the delayed senescence of plants. In conclusion, based on the results of the present study, we hereby propose a model that might be involved in the stay-green phenotype of tasg1 (Fig. 9). The cisZOGT1 gene has an important role in regulating wheat leaf senescence by regulating CK and N metabolism. At the same time, CK and N metabolism involved in delayed flag leaf senescence of tasg1 may be by a feedback pattern. Conflicts of interest The authors have no conflicts of interest to declare. Author contribution Conceived and designed the experiments: W. Wang, J. J. Wu. Performed the experiments: W.Q. Wang, Q.Q. Hao, Q.X. Li, F.J. Chen. Analyzed the data: W.Q. Wang, Q.Q. Hao. Contributed reagents/materials/analysis tools: F. Ni, Y. Wang, D.L. Fu, J.J. Wu. Wrote the paper: W.Q. Wang, Q.Q. Hao. Proof read and final approval: W. Wang.
Fig. 9. Proposed model for stay-green phenotype in tasg1.
the tasg1 plants was lower than that in the WT plants [1]. Previous studies have shown that N starvation induced leaf senescence in winter oilseed rape [35]. Studies have also shown that increased CK levels significantly delayed the senescence of transgenic maize plants [6]. However, the relationship between CK and N in regulating plant senescence was not clear. To investigate this relationship, WT and tasg1 plants were treated with NH4NO3 and/or lovastatin. When treated with NH4NO3, the WT and tasg1 plants exhibited a delayed senescence phenotype (Fig. 6A–C). Furthermore, not only the total N content, but also IPA, ZR, and DHZR contents were significantly increased in the WT and tasg1 plants (Fig. 6D–G). At the same time, the expression levels of CK biosynthesis related genes were significantly higher in the WT and tasg1 plants (Fig. S4A–C). The data presented in Figs. 6, S3, and S4 suggested that maybe the N metabolism functioned in leaf senescence through the regulation of CKs metabolism. Then, when treated with lovastatin, the tasg1 plants exhibited the premature cell death phenotype (Fig. 7A–C). Not only IPA, ZR, and DHZR, but also N contents were significantly decreased in the tasg1 plants (Fig. 7D–G). However, when treated with lovastatin plus NH4NO3, the premature cell death phenotype of tasg1 plants was partly recovered (Fig. 7A–C), which was consistent with the increased IPA, ZR, and DHZR, and total N contents (Fig. 7D–G). Morever, when the WT and tasg1 plants were treated with BAP, the WT and tasg1 plants exhibited a delayed senescence phenotype (Fig. S7A). At the same time, not only N content but also N metabolism genes expression levels were significantly higher in WT and tasg1 plants (Fig. S7B-H). The data presented in Figs. 7, S3, S5 and S7 suggested that CKs metabolism might regulate N metabolism, and then functioned in leaf senescence. Taken together, the results in Figs. 6,7, S3, S4, and S5 suggested that the feedback of CK on N content was involved in regulating leaf senescence in tasg1 plants.
Acknowledgements We thank Professor Jorge Dubcovsky at University of California Davis for providing EMS mutants of durum wheat Kronos. This work was supported by the National Natural Science Foundation of China (No. 31370304), the National Key Research and Development Program of China (2016YFD0101004) and by Funds of Shandong “Double Tops’’ Program. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.plantsci.2018.10.024. References [1] W.Q. Wang, Q.Q. Hao, F.X. Tian, W. Wang, The stay-green phenotype of wheat mutant tasg1 is associated with altered cytokinin metabolism, Plant Cell Rep. 35 (2016) 585–599. [2] G. Spano, N.D.I. Fonzo, C. Perrotta, Physiological characterization of ‘stay green’ mutants in durum wheat, J. Exp. Bot. 54 (2003) 1415–1420. [3] S. Ha, R. Vankova, K. Yamaguchi-Shinozaki, K. Shinozaki, L.P. Tran, Cytokinins: metabolism and function in plant adaptation to environmental stresses, Trends Plant Sci. 17 (2012) 172–179. [4] J. Van Staden, E.L. Cook, L.D. Noodèn, Cytokinins and senescence, in: L.D. Noodèn, A.C. Leopold (Eds.), Senescence and Aging in Plants, Academic Press, San Diego, 1988, pp. 281–328. [5] E. Taverner, D.S. Letham, J. Wang, E. Cornish, D.A. Willcocks, Influence of ethylene on cytokinin metabolism in relation to Petunia corolla senescence, Phytochemistry. 51 (1999) 341–347. [6] P.R.H. Robson, I.S. Donnison, K. Wang, Leaf senescence is delayed in maize expressing the Agrobacterium IPT gene under the control of a novel maize senescenceenhanced promoter, Plant Biotechnol. J. 2 (2004) 101–112. [7] M.L. Cao, Performance of autoregulatory senescence-inhibition in rice, Hum. Agric. Agric. Sci. Technol. News l2 (2001) 17–24. [8] Y.J. Lin, M.L. Cao, C.G. Xu, H. Chen, J. Wei, Q.F. Zhang, Cultivating rice with delaying leaf senescence by P-SAG12-IPT gene transformation, Acta Bot. Sin. 44 (2002) 1333–1338. [9] C. Masclaux, I. Quillere, A. Gallais, B. Hirel, The challenge of remobilization in plant nitrogen economy. A survey of physio-agronomic and molecular approaches, Ann. Appl. Biol. 138 (2001) 69–81. [10] A.K. Borrell, G.L. Hammer, E. Van Oosterom, Stay-green: a consequence of the balance between supply and demand for nitrogen during grain filling, Ann. Appl. Biol. 138 (2001) 91–95. [11] E.J. Van Oosterom, A.K. Borrell, S.C. Chapman, I.J. Broad, G.L. Hammer, Functional dynamics of the nitrogen balance of sorghum: I. N demand of vegetative plant parts, Field Crop Res. 115 (2010) 19–28. [12] P. Martre, J.R. Porter, P.D. Jamieson, E. Triboi, Modelling grain nitro-gen accumulation and protein composition to understand sink/source regulations of nitrogen remobilization for wheat, Plant Physiol. 133 (2003) 1959–1967. [13] O. Gaju, V. Allard, P. Martre, J.L. Gouis, D. Moreau, M. Bogard, Nitrogen
4.2. cisZOGT1-regulated CK and N content in the delayed senescence of plants Glycosylation plays an important role in plant growth and development; O-glycosylation, is thought to have an effect on the balance of hormones [36]. It is assumed that CK O-glucosides represented reversibly inactivated storage forms [17]. The cisZOGTs preferentially catalyzed the O-glucosylation of cis-zeatin in vitro [37]. Transgenic maize over-expressing the cisZOGT1 gene presented the CK deficient phenotype, although it also showed the delayed senescence phenotype [17]. In this study, the knock-out lines of the cisZOGT1 gene, mutant 2885-1, 2885-2, and 2885-3, exhibited the delayed senescence phenotype at the late filling stage (Fig. 8A). Moreover, the total N, ZR, and 78
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Research Article
The involvement of cytokinin and nitrogen metabolism in delayed flag leaf senescence in a wheat stay-green mutant, tasg1
T
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Wenqiang Wanga,b, ,1, Qunqun Haoc,d,1, Wenlong Wangb,1, Qinxue Lib, Fengjuan Chend, Fei Nid, ⁎ ⁎ Yong Wangb, Daolin Fuc, Jiajie Wud, , Wei Wangb, a
College of Life Sciences, Zaozhuang University, Zaozhuang, 277000, China State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, 271018, Shandong, China c Department of Plant Sciences, University of Idaho, Moscow, ID, 83844, USA d State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai’an, 271018, Shandong, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Leaf senescence CK metabolism N metabolism RNA-seq Stay-green Wheat
In the present study on a wheat stay-green mutant, tasg1, we found that its delayed senescence at the late filling stage was related to the high cytokinin (CK) and N contents. RNA sequencing suggested that several genes may be responsible for the different senescence processes between wild-type (WT) and tasg1 plants. WT and tasg1 seedlings were treated with NH4NO3, lovastatin, and 6-benzylaminopurine (BAP), and the results suggested that the feedback of CK with N content regulated the leaf senescence in the tasg1 plants. Furthermore, a knock-out of the candidate gene cisZOGT1 (catalytic O-glucosylation in cis-zeatin) in the wheat mutant pool ‘Kronos’ exhibited delayed senescence at the late grain filling stage. Overall, our results suggested the cisZOGT1 gene has an important role in regulating wheat leaf senescence by regulating CK and N metabolism. At the same time, CK and N metabolism involved in delayed flag leaf senescence of tasg1 may be by a feedback pattern.
1. Introduction Leaf senescence is generally the last stage of leaf development, during which a fully-expanded leaf progresses to its death. Plant senescence is an internally programmed degenerative process, and premature leaf senescence can result in a low grain yield [1]. Compared to WT plants, stay-green or non-yellowing mutants of various plant species have been reported to maintain the greenness of their leaves during senescence for a longer time, and are ideal materials for studying the mechanisms underlying plant senescence [2], which, to date, remains unclear. Cytokinins (CKs), which are widespread in nature, can be distinguished as isopentenyladenine (iP)-, trans-zeatin (tZ)-, cis-zeatin (cZ)- or dihydrozeatin-type derivatives. The rate-limiting step of isoprenoid CK biosynthesis is catalyzed by isopentenyltransferases (IPTs) [3]. The initiation of senescence involves alterations in the levels of CK [4]. Exogenous application of 6-benzylaminopurine (6-BA, a synthetic CK), at concentrations ranging from 2 to 200 μM, was reported to almost completely prevent petunia corolla senescence [5]. Transgenic expression of the IPT gene in maize, which encodes an isopentenyl
transferase, increased the CK levels and resulted in delayed senescence [6]. Moreover, senescence-regulated expression of IPT in rice was observed to delay the senescence of leaves, with regard to both the chlorophyll degradation and photosynthetic capacity [7,8]. The genetic control of N remobilization is linked to the regulation of leaf senescence [9]. Stay-green, the ability to retain green areas in the grain during filling, has been found to be related to the supply–demand balance of N during grain filling in Sorghum [10,11]. Modifying the N remobilization efficiency to delay senescence in feed wheat cultivars could favor a longer stage of active photosynthesis during grain filling, and might result in a higher grain yield [12]. Cultivars with prolonged N uptake under N-deficient conditions characteristically have delayed senescence in older leaves [13]. The side-chain modifying steps of CK, such as O-glycosylation, are expected to have a direct bearing on CK-mediated processes [14]. Veach et al. [15] reported that cisZOGT1 catalyzed O-glucosylation in the N6-sidechain of cis-zeatin. Compared to the highly active transzeatin, cis-zeatin is generally regarded as a CK with little or no activity [16]. Rodó et al. [17] also put forward that the characteristics of maize overexpressing the cisZOGT1 gene resembled those of CK-deficient
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Corresponding authors. Corresponding author at: College of Life Sciences, Zaozhuang University, Zaozhuang, 277000, China. E-mail addresses: [email protected] (W. Wang), [email protected] (J. Wu), [email protected] (W. Wang). 1 These authors contributed equally to this work. ⁎⁎
https://doi.org/10.1016/j.plantsci.2018.10.024 Received 30 August 2018; Received in revised form 18 October 2018; Accepted 31 October 2018 Available online 02 November 2018 0168-9452/ © 2018 Elsevier B.V. All rights reserved.
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measured according to the method of Lillo [21].
plants, whereas, a delayed senescence phenotype was exhibited in transgenic maize. Although some studies suggested possible roles for cis-zeatin, its physiological significance remains unclear. A wheat stay-green mutant, tasg1, was previously generated through mutagen breeding in our laboratory with the application of ethyl methane sulfonate (EMS) to the WT of the ‘HeSheng2′ cultivar. Our studies revealed that the stay-green phenotype of tasg1 was associated with altered CK metabolism [1]. Moreover, sucrose metabolism, regulated by CKs, was also involved in the delayed senescence of tasg1 [18]. In the present study, using RNA-seq, we found differentially expressed genes (DEGs) related to CK and N metabolism between WT and tasg1. Then, we used tasg1, 2885 (a durum wheat mutant) to determine two details of CK and N metabolism in the delayed senescence phenotype. 1) The feedback between CK and N metabolism in the delayed senescence of tasg1; 2) The regulation of wheat leaf senescence by cisZOGT1, and the involvement of CK and N metabolisms in cisZOGT1-regulated senescence.
2.6. Determination of free amino acid content The methods for the extraction and measurement of free amino acids were modified according to Li et al. [22]. The external standards for free amino acid were procured from Agilent Technologies (Waldbronn, Germany). 2.7. Quantitative reverse transcription PCR analysis Total RNA was extracted from the second leaf with TransZol (TransGen Biotech, China), and treated with DNase I (RNase-free, Promega). qRT-PCR was carried out in a 25 μL reaction volume containing 2× TransStart Top Green q-PCR SuperMix (TRANS, China). Quantitative analysis was performed using the Bio Rad CFX Manager system. This method normalizes the expression of a specific gene versus a control reference with the formula 2−△△CT. Actin and tubulin were evaluated as the control genes. Information on the genes analyzed is listed in Table S1.
2. Materials and methods 2.1. Field experiments
2.8. RNA sequencing and data analysis Field experiments were carried out at the farm of Shandong Agricultural University, China. The growth and development of the wheat plants were managed according to conventional agricultural techniques. Six interspersed plots (4 m2) were selected randomly in the field. Each plot was used for sample collection. The experiment was conducted at least in triplicate.
We collected two types of samples from the WT and tasg1 plants at the filling stage (28 d). Three biological replicates were used for each tissue genotype for the RNA sequencing. Total RNA was extracted from each sample using TRIzol reagent, following the manufacturer’s specifications (Invitrogen). The quality and quantity of each RNA sample was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). The mRNAs were isolated from the total RNA using Dynabeads mRNA DIRECT Kit (Invitrogen) and were fragmented into short fragments using a fragmentation buffer. Using these short fragments as templates, random primers, and a SuperScript double-stranded cDNA synthesis kit (Invitrogen), double-stranded cDNA was synthesized. Ligated fragments were then generated by a series of reactions that included purification of the PCR products, end repair, dA-tailing, and ligation of the Illumina adapters. After agarose gel electrophoresis, suitable fragments were selected for PCR amplification. The final library was evaluated by quantitative RT-PCR with a StepOne Plus RealTime PCR system (Applied Biosystems, Foster City, CA, USA). Sequencing reactions were performed on the Illumina HiSeq platform (PE 150) by the Berry Genomics Company (Beijing, China). Functional annotation and enrichment analysis of the differentially expressed genes (DEGs) was conducted using the Gene Ontology (GO) database (http://www.geneontology.org/). Information on the gene annotation is listed in Table S2.
2.2. Laboratory experiments WT and tasg1 seeds were germinated on filter paper, moistened with water for 24 h at 25 ± 1 °C after being sterilized with 0.2% sodium hypochlorite. Thereafter, the seeds were grown at 25 °C under a 16 h light/8 h dark illumination cycle (light intensity 300 μmol m−2 s-1) and 70% relative humidity. A nutrientless hydroponic system was used to induce leaf senescence in the wheat seedlings. The tasg1 plants showed a significantly delayed senescence phenotype in the late growth stage (11 d) compared to the WT plants. For BAP treatment, the wheat seedlings were sprayed by a 100 μM BAP solution (Sigma) with 0.02% (v/v) Tween-20. 2.3. Measurement of CKs The methods for the extraction and purification of CKs were modified according to Wang et al. [1]. The antigens, isopentenyl adenine (IPA), zeatin riboside (ZR), dihydrozeatin riboside (DHZR), and GA4 used in ELISA, and the mouse monoclonal antibodies against them were produced at the Phytohormones Research Institute of China Agricultural University.
2.9. Histochemical detection To visualize the dead cells, the detached leaves were stained with trypan blue (TB) [23]. The leaves were detached and submerged in 0.05% lactophenol–TB solution (0.05% TB, 25% [w/v] lactic acid, 25% water-saturated phenol, and 50% ethanol) at 70 °C for 10 min, and then heated in boiling water for 5 min and left overnight for staining. Destaining was subsequently performed in a 2.5 g μL−1 chloral hydrate solution for 3 d to reduce the background staining.
2.4. Measurement of the N content The leaf N content was determined according to the Kjeldahl method [19]. 2.5. Determination of NR, NiR, and GS activity
2.10. Chlorophyll content To determine the activity of different enzymes, leaves (0.1 g) were homogenized in 100 mM potassium phosphate buffer (pH 7.4), containing 7.5 mM cysteine, 1 mM EDTA, and 1.5% (w/v) casein using a chilled mortar and pestle. The homogenate was centrifuged at 20,000 × g for 15 min at 4 °C. The activities of Nitrate reductase (NR) and Nitrite reductase (NiR) were determined according to the method of Plett et al. [20]. The activity of Glutamine synthetase (GS) was
The chlorophyll contents were determined based on the method of Lichtenthaler [24]. 2.11. Statistical analysis All analyses were conducted, at least, in triplicate. The IBM SPSS 71
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Fig. 1. Different time-course dynamics during the filling stage between the wild-type (WT) and tasg1 plants. The parameters include the phenotype at different daypoints after flowering (A); the relative water content (B); the phenotype of flag leaves (28 d) (C); and the IPA (D), ZR (E), DHZR (F), and N (G) contents. Values are means ± SD based on thirty replicates. FW, fresh weight. DW, dry weight. Error bars indicate standard deviations.
plants (Fig. S1D). We also determined the expression of three senescence-regulated genes, namely, TaSAG1 (Fig. 2A), TaSAG2 (Fig. 2B), and TaSAG4 (Fig. 2C). Their expressions in the tasg1 plants were down-regulated compared to those in the WT plants. However, the expressions of CK biosynthesis-regulated genes, TaIPT2 (Fig. 2D), TaIPT3 (Fig. 2E), and TaIPT8 (Fig. 2F), were significantly higher in the tasg1 plants than in the WT plants. Furthermore, the expressions of N transport-regulated genes, TaNRT1.1 (Fig. 2G), TaNRT1.3 (Fig. 2H), and TaNRT1.4, (Fig. 2I) and amino acid synthesis-regulated genes, TaAlaAT (Fig. 2J), TaGS1 (Fig. 2K), and TaGS2 (Fig. 2L), were also significantly higher in the tasg1 plants than in the WT plants. These results suggest that both CK and N metabolism may be involved in the delayed senescence of tasg1, compared to that in its WT plant.
Statistics program was used to perform the statistical analyses. All comparisons were analyzed using factorial ANOVAs. Differences between the means among the wheat lines or treatments were compared using Duncan’s multiple range tests, at 0.05 probability levels. 3. Results 3.1. Dynamic delayed senescence phenotype of tasg1 plants at the filling stage We observed the dynamic phenotype throughout the filling stage of WT and tasg1 plants. We found that the senescence became evident at 21 d, after flowering in the WT plants (Fig. 1A). However, the senescence of tasg1 plants did not appear until day 24 of flowering (data not shown). Compared to the WT plants, the tasg1 plants had obviously delayed senescence at the late stages (28 d, 32 d, respectively) (Fig. 1A). Moreover, at the early stage (before 21 d), there was no significant difference in the relative water content between the WT and tasg1 plants, but at the late filling stage, it was 12.6% and 20.4% higher on days 28 and 32, respectively, in tasg1 than in WT (Fig. 1B). Based on the differences in the senescence phenotype between tasg1 and WT as shown in Fig. 1A, and the relative water content as shown in Fig. 1B, we selected the flag leaf at 28 d as the experimental material for the subsequent study (Fig. 1C).
3.3. RNA-seq analysis of the differential expression of genes related to CK and N metabolisms between WT and tasg1 plants To systematically analyze the contribution of CK and N metabolism to delayed leaf senescence in tasg1 plants, we performed RNA-seq using the flag leaves of WT and tasg1 plants, at 28 d after flowering. Compared to the WT samples, the transcripts in tasg1 samples had 8373 DEGs, with their expression differing by two-fold or more; of these, 3906 genes were down-regulated and 4467 genes were up-regulated (Fig. 3A and B, Table S2). To validate these DEGs, a qRT-PCR assay was performed for 19 randomly selected genes. The results shown in Fig. 3C showed the consistency between the RNA-seq and qRT-PCR data, indicating the credibility of the RNA-seq data. A total of 526 transcription factors (TFs) were differentially expressed, of which 238 were downregulated and 288 were up-regulated in the tasg1 plants compared to their expression levels in the WT plants (Fig. S2). The expression levels of the C2C2-YABBY family members were down-regulated, whereas those of the mTERF, Tify, and HSF family members were up-regulated. The expressions of some members in transcription factors families, such as MYB, bHLH, NAC, WRKY, and AP2/EREBP families, were either down-regulated or up-regulated. GO analysis was performed for the DEGs using the GO database,
3.2. Changes in CK and N metabolisms in tasg1 at the filling stage We measured the contents of CKs, including isopentenyl adenine (IPA), zeatin riboside (ZR), and dihydrozeatin riboside (DHZR), in tasg1 and WT at the filling stage. The contents of IPA, ZR, and DHZR were 53.8%, 17.6%, and 38.2% higher in tasg1 plants than in WT, respectively (Fig. 1D–F). Furthermore, the total N content in the tasg1 plants was about two-fold that in the WT plants (Fig. 1G). Simultaneously, the activities of NR, NiR, and GS showed a trend similar to that of N content; they were approximately 2–3-fold in the tasg1 plants compared to those in the WT plants (Fig. S1A–C). The free amino acid concentrations in the tasg1 plants were also significantly higher than those in the WT 72
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Fig. 2. Relative expression of genes related to senescence, CK biosynthesis, N transport, and amino acid synthesis, including TaSAG1 (A), TaSAG2 (B), TaSAG4 (C), TaIPT2 (D), TaIPT3 (E), TaIPT8 (F), TaNRT1.1 (G), TaNRT1.3 (H), TaNRT1.4 (I), TaAlaAT (J), TaGS1 (K), and TaGS2 (L) in the flag leaves (28 d) of wild-type (WT) and tasg1 plants at the filling stage. Values are means ± SD based on six replicates. Error bars indicate standard deviations.
Fig. 3. RNA-seq analysis of the transcriptome of flag leaves (28 d). The parameters include differentially expressed genes (DEGs) (A), changes in the gene expression profile of flag leaves (28 d) between wild-type (WT) and tasg1 plants (B), confirmation of RNA-seq by qRT-PCR data (C). Values are means ± SD based on three replicates. 73
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Fig. 4. Histograms of gene ontology annotation of CK metabolism in flag leaves (28 d) and wild-type (WT) and tasg1 plants at the filling stage. The parameters include the percentage of DEGs in biological process (A), biological process (B), the percentage of DEGs in molecular function (C), and molecular function (D). Values are mean ± SD based on three replicates.
the nitrogen fixation, ammonia assimilation cycle, cellular amino acid metabolic process, glutathione biosynthetic process, glutamine biosynthetic process, and glutamate metabolic process (Fig. 5A and B; Table S2). Moreover, for the molecular functions, among the downregulated genes, those encoding the cytokinin dehydrogenase and oxidoreductase were clearly over-represented in this category (Fig. 5C; Table S2). However, among the up-regulated genes, those encoding glutamate-ammonia ligase, ammonia ligase, acid-ammonia ligase, and glutathione synthase were clearly over-represented in the molecular function category (Fig. 5D; Table S2). Overall, the RNA-seq results shown in Figs. 3 and 5 also suggest that both CK and N metabolism may be involved in the delayed senescence of the tasg1 seen in Figs.1 and 2.
which provides information on the annotation of biological processes and molecular functions (http://www.geneontology.org/). Specifically, we analyzed the biological processes and molecular functions of CKs. For the biological process, obvious over-representation was observed for CKs metabolic processes, including cytokinin metabolic process, cellular hormone metabolic process, cytokinin catabolic process, and hormone metabolic process (Fig. 4A and B; Table S2). Moreover, for the molecular functions, among the down-regulated genes, a large number of genes encoding the cytokinin dehydrogenase and oxidoreductase were clearly over-represented in this category (Fig. 4C; Table S2). However, among the up-regulated genes, only a few genes encoding the cytokinin dehydrogenase and oxidoreductase were clearly over-represented in the molecular function category (Fig. 4D; Table S2) Furthermore, we also analyzed the biological processes and molecular functions of N metabolism. For the biological process, obvious over-representation was observed for N metabolic processes, including 74
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Fig. 5. Histograms of gene ontology annotation of N metabolism in flag leaves (28 d) of WT and tasg1 plants at the filling stage. The parameters include the percentage of DEGs in biological process (A), biological process (B), the percentage of DEGs in molecular function (C), and molecular function (D). Values are mean ± SD based on three replicates.
with NH4NO3, the activities of NR, NiR, and GS were significantly higher than those in the untreated plants (Fig. S3A–C). The expression levels of the nine genes related to CK biosynthesis, N transport, and amino acid synthesis were also determined. Under normal conditions, the expression levels of TaIPT2 (Fig. S4A), TaIPT3 (Fig. S4B), TaIPT8 (Fig. S4C), TaNRT1.1 (Fig. S4D), TaNRT1.3 (Fig. S4E), TaNRT1.4 (Fig. S4F), TaAlaAT (Fig. S4G), TaGS1 (Fig. S4H), and TaGS2 (Fig. S4I) were significantly higher in the tasg1 plants than in the WT plants. Furthermore, in the NH4NO3-treated WT and tasg1 plants, the expression levels of genes involved in N transport (TaNRT1.1, TaNRT1.3, and TaNRT1.4) and amino acid synthesis (TaAlaAT, TaGS1, and TaGS2), as well as those involved in CK biosynthesis (TaIPT2, TaIPT3, and TaIPT8) were significantly up-regulated in the WT and tasg1 plants compared to those in the untreated plants. These results suggest that CK metabolism was regulated by N content and/or N metabolism, which in turn affect wheat leaf senescence. To further substantiate the role of CKs in regulating leaf senescence, and to understand the involvement of CKs in N metabolism, the tasg1 plants were treated with 40 μM lovastatin (The seedlings were cultivated in a 40 μM lovastatin solution when they were grown to 7 days), which is a specific inhibitor of CK biosynthesis, and with 40 μM lovastatin along with 0.1 M NH4NO3. Under normal growth conditions, the tasg1 plants exhibited a delayed senescence phenotype compared to that of WT plants (Fig. 7A). The TB staining indicated that cell death
3.4. The feedback between CK and N content in the regulation of senescence in tasg1 plants To understand the involvement between the metabolisms of CK and N in the regulation of leaf senescence in tasg1, we externally controlled the N content and regulated the CK metabolism. To induce leaf senescence, we used two-week-old wheat seedlings in a nutrientless hydroponic system [1]. To control the N contents, WT and tasg1 plants were treated with 0.1 M NH4NO3. The seedlings were cultivated in a 0.1 M NH4NO3 solution when they were grown to 7 days. Since leaf senescence is usually accompanied by cell death, we examined the effect of NH4NO3 on cell death by Trypan Blue (TB) staining because the function of TB is to dye dead cells [25]. The WT and tasg1 plants exhibited a delayed senescence phenotype, and had reduced cell death (Fig. 6A and B). Moreover, the chlorophyll contents were also significantly increased in the WT and tasg1 plants when treated with NH4NO3 (Fig. 6C). We also measured the contents of the different CKs (IPA, ZR, and DHZR) and total N in the WT and tasg1 plants. The NH4NO3 treatment significantly increased the N content (Fig. 6G), as well as that of IPA (Fig. 6D), ZR (Fig. 6E), and DHZR (Fig. 6F) in the WT and tasg1 plants. We also examined the activity of NR, NiR, and GS, which are related to N metabolism (Fig. S3). Under normal conditions, the activities of NR, NiR, and GS in the tasg1 plants were significantly higher than those in the WT plants. However, when the WT and tasg1 plants were treated
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Fig. 6. Effect of NH4NO3 on wild-type (WT) and tasg1 seedling leaves at 11 d of growth. The parameters include the phenotype (A), trypan blue staining (B), chlorophyll content (C), isopentenyl adenine (IPA) content (D), ZR content (E), dihydrozeatin ribosideDHZR content (F), and N content (G). Values are means ± SD based on three replicates. FW, fresh weight. DW, dry weight. Error bars indicate standard deviations.
We further examined the expression of the genes related to CK biosynthesis, N transport, and amino acid synthesis (Fig. S5). Under normal conditions, the expression levels of TaIPT2 (Fig. S5 A), TaIPT3 (Fig. S5B), TaIPT8 (Fig. S5C), TaNRT1.1 (Fig. S5D), TaNRT1.3 (Fig. S5E), TaNRT1.4 (Fig. S5F), TaAlaAT (Fig. S5G), TaGS1 (Fig. S5H), and TaGS2 (Fig. S5I) were significantly higher in the tasg1 plants than in the WT plants. When treated with lovastatin, the expression levels of the genes involved in CK biosynthesis (TaIPT2, TaIPT3, and TaIPT8), N transport (TaNRT1.1, TaNRT1.3, and TaNRT1.4), and amino acid synthesis (TaAlaAT, TaGS1, and TaGS2) were significantly decreased in the tasg1 plants (Fig. S5). However, when treated with lovastatin plus NH4NO3, the expression of these genes was up-regulated (Fig. S5). We also examined the expression of the genes related to CK signaling (Fig. S6). However, when treated with lovastatin plus NH4NO3, the ARR6 (type-A response regulator) (Fig. S6A) was expressed at a lower level in tasg1 plants. In contrast, the expression of ARR18 (type-B response regulator) (Fig. S6B) was up-regulated in tasg1 plants. This result showed that application of N not only regulated CK biosynthesis pathway but also CK signaling pathway. Moreover, when the WT and
was obviously reduced in the tasg1 plants (Fig. 7B). When the tasg1 plants were treated with lovastatin, they exhibited a premature phenotype and increased cell death (Fig. 7A and B). However, application of NH4NO3 partially restored the premature senescence phenotype and reduced cell death of tasg1 with lovastatin treatment (Fig. 7A, B). The chlorophyll content corroborated with the phenotypic information (Fig. 7C). In addition, we measured the contents of the different CKs and total N levels. Under normal conditions, the IPA content (Fig. 7D), ZR (Fig. 7E), DHZR (Fig. 7F), and total N (Fig. 7G) in the tasg1 plants were approximately 1.06-, 1.83-, 1.86-, and 1.29-fold higher than those in the WT plants, respectively. However, when treated with lovastatin, their contents were significantly decreased (Fig. 7G) in the tasg1 plants. Furthermore, when treated with lovastatin plus NH4NO3, the contents of different CKs and total N were significantly increased in the tasg1 plants (Fig. 7D–G). We also examined the activities of NR, NiR, and GS in the WT and tasg1 plants (Fig. S3). When treated with lovastatin, their activities were significantly decreased in the tasg1 plants. However, when treated with lovastatin plus NH4NO3, their activities slightly increased in the tasg1 plants (Fig. S3D–F).
Fig. 7. Effect of lovastatin (lov, CKs inhibitor), and lovastatin plus NH4NO3 on wild-type (WT) and tasg1 seedling leaves at 11 d of growth. The parameters include the phenotype (A), trypan blue staining (B), chlorophyll content (C), isopentenyl adenine (IPA) content (D), ZR content (E), dihydrozeatin ribosideDHZR content (F), and N content (G). Values are means ± SD based on three replicates. FW, fresh weight. DW, dry weight. Error bars indicate standard deviations. 76
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this study, tasg1 plants exhibited a markedly delayed senescence phenotype at the late filling stage (Fig. 1A). N is an essential macronutrient for plant growth and development. The main symptom of N deficiency in plants is leaf senescence, which is a result of pigment loss, protein degradation, and lipid peroxidation [29]. Many studies have shown that N also plays an important role in the regulation of plant senescence [30,31]. In the present study, in addition to the contents of three types of CK, the N content in the flag leaves of tasg1 plants was also significantly higher than that in the WT plants at the late filling stage (Fig. 1C–G). Nitrate reductase, NiR, and GS are the three key enzymes involved in protein synthesis, and contribute to the growth of plants [20]. The activities of NR, NiR, and GS indicate that tasg1 plants have strong N assimilation and NH4+ transport capacities (Fig. S1). Studies have shown that senescence-associated genes (SAGs) are transcriptionally up-regulated during senescence [32]. The expression of TaSAGs further advanced the delayed senescence phenotype in the tasg1 plants (Fig. 2A–C). The expression of mRNAs related to CK biosynthesis, N transport, and amino acid synthesis indicates that both the CK and N metabolisms were changed in the flag leaves of tasg1 plants compared with those in the WT (Fig. 2D–L). RNA-seq was used to analyze the different expression of genes involved in CK and N metabolisms between tasg1 and its WT. Compared to WT plants, only a few genes encoding the cytokinin dehydrogenase and oxidoreductase were clearly over-represented in tasg1 plants in the molecular function category (Fig. 4C and D). The GO analysis of CK metabolism also supported the data of the increased CKs in tasg1 plants at the late filling stage, which can regulate the senescence of plants (Figs. 3, 4; Table S2). Notably, the GO analysis of N metabolism further revealed the difference in N metabolism between tasg1 and WT plants. Besides, a large number of genes encoding the N synthesis and transport in tasg1 at the late filling stage were examined (Figs. 3, 5; Table S2). From the results in Figs. 1–5, we suggest that CK and N metabolisms affect the delayed senescence of tasg1 plants. Transcriptional activators and repressors play crucial roles in mediating the signalling pathway of leaf senescence [25]. Previous studies have identified a few transcriptional regulators of H2O2-induced leaf senescence, including HSF, AP2/EREBP, and NAC [33,34]. Intriguingly, the expression levels of several TFs, including HSF and AP2/EREBP, were enriched in the up-regulated group (Fig. S2). These results imply that H2O2 scavenging might be involved in delayed leaf senescence in tasg1 plants. Our previous study also indicated that the H2O2 content in
tasg1 plants were treated with BAP, the WT and tasg1 plants exhibited a delayed senescence phenotype (Fig. S7A). At the same time, not only N content but also the expression levels of N metabolism genes (TaNRT1.1, TaNRT1.3, TaNRT1.4, TaAlaAT, TaGS1, and TaGS2) were significantly higher in WT and tasg1 plants (Fig. S7B-H). Taken together, these results suggested that CKs metabolism was also involved in N metabolism in regulating wheat leaf senescence. 3.5. The role of cisZOGT1 in delayed plant senescence We analyzed the DEGs for cytokinin metabolic pathways (about 8fold) between WT and tasg1. cisZOG1 (Traes_2 AL_21065D298) and CKX1 (Traes_3 AS_BEB2E9AA9) were screened in this study. The function of CK dehydrogenases1 (CKX1) gene has been widely reported in previous studies. CK degradation requires CKXs, which remove CK unsaturated isoprenyl side chains [26]. At the same time, overexpression of CKX1 leads to stronger CK deficient phenotype than that of other CKX genes [27]. Therefore, we focus on cisZOGT1 gene in this study. RNA-seq data showed that the expression of cisZOGT1 in tasg1 was significantly lower than that of WT (about 14%). This cisZOGT1 gene encoded a cis-zeatin O-glucosyltransferase 1. The results of qRTPCR further proved the lower expression of the cisZOGT1 gene in tasg1 than in WT (Fig. S8). Then, a knock-out of the cisZOGT1-B locus was screened from the durum wheat mutant pool ‘Kronos’ (Fig. S9). Therein, mutant 2885 lines, including 2885-1, 2885-2, and 2885-3 exhibited delayed leaf senescence (Fig. 8A). Moreover, the chlorophyll content in flag leaves of 2885 lines was significantly higher than that in WT at the late filling stage (Fig. 8B). The total N (Fig. 8C), ZR (Fig. 8E), and DHZR (Fig. 8F) contents in the mutant 2885 plants were also approximately 1.29-, 1.32-, and 1.11-fold higher than those in the WT, but no obvious differences were observed in IPA (Fig. 8D). 4. Discussion 4.1. CK and N metabolisms affect delayed leaf senescence in tasg1 plants Leaf senescence is a critical step in the life cycle of an annual plant. During the process, the apparent visual characteristics of leaf senescence are the loss of chlorophyll and eventual abscission [28]. In a previous report, we demonstrated that the altered CK metabolism was involved in the stay-green phenotype of the wheat mutant, tasg1 [1]. In
Fig. 8. The phenotype of WT and mutant 2885 during the late growth stage. The parameters include the phenotype (A), the chlorophyll content (B), N (C), isopentenyl adenine (IPA) (D), ZR (E), and dihydrozeatin ribosideDHZR (F). Values are means ± SD based on three replicates. FW, fresh weight. DW, dry weight. Error bars indicate standard deviations. 77
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DHZR contents in the mutant 2885 lines were higher than those in their respective WT plants (Fig. 8C, E, and F). These results reinforce the opinion that CK and N levels regulated by cisZOGT1 were important to the delayed senescence of plants. In conclusion, based on the results of the present study, we hereby propose a model that might be involved in the stay-green phenotype of tasg1 (Fig. 9). The cisZOGT1 gene has an important role in regulating wheat leaf senescence by regulating CK and N metabolism. At the same time, CK and N metabolism involved in delayed flag leaf senescence of tasg1 may be by a feedback pattern. Conflicts of interest The authors have no conflicts of interest to declare. Author contribution Conceived and designed the experiments: W. Wang, J. J. Wu. Performed the experiments: W.Q. Wang, Q.Q. Hao, Q.X. Li, F.J. Chen. Analyzed the data: W.Q. Wang, Q.Q. Hao. Contributed reagents/materials/analysis tools: F. Ni, Y. Wang, D.L. Fu, J.J. Wu. Wrote the paper: W.Q. Wang, Q.Q. Hao. Proof read and final approval: W. Wang.
Fig. 9. Proposed model for stay-green phenotype in tasg1.
the tasg1 plants was lower than that in the WT plants [1]. Previous studies have shown that N starvation induced leaf senescence in winter oilseed rape [35]. Studies have also shown that increased CK levels significantly delayed the senescence of transgenic maize plants [6]. However, the relationship between CK and N in regulating plant senescence was not clear. To investigate this relationship, WT and tasg1 plants were treated with NH4NO3 and/or lovastatin. When treated with NH4NO3, the WT and tasg1 plants exhibited a delayed senescence phenotype (Fig. 6A–C). Furthermore, not only the total N content, but also IPA, ZR, and DHZR contents were significantly increased in the WT and tasg1 plants (Fig. 6D–G). At the same time, the expression levels of CK biosynthesis related genes were significantly higher in the WT and tasg1 plants (Fig. S4A–C). The data presented in Figs. 6, S3, and S4 suggested that maybe the N metabolism functioned in leaf senescence through the regulation of CKs metabolism. Then, when treated with lovastatin, the tasg1 plants exhibited the premature cell death phenotype (Fig. 7A–C). Not only IPA, ZR, and DHZR, but also N contents were significantly decreased in the tasg1 plants (Fig. 7D–G). However, when treated with lovastatin plus NH4NO3, the premature cell death phenotype of tasg1 plants was partly recovered (Fig. 7A–C), which was consistent with the increased IPA, ZR, and DHZR, and total N contents (Fig. 7D–G). Morever, when the WT and tasg1 plants were treated with BAP, the WT and tasg1 plants exhibited a delayed senescence phenotype (Fig. S7A). At the same time, not only N content but also N metabolism genes expression levels were significantly higher in WT and tasg1 plants (Fig. S7B-H). The data presented in Figs. 7, S3, S5 and S7 suggested that CKs metabolism might regulate N metabolism, and then functioned in leaf senescence. Taken together, the results in Figs. 6,7, S3, S4, and S5 suggested that the feedback of CK on N content was involved in regulating leaf senescence in tasg1 plants.
Acknowledgements We thank Professor Jorge Dubcovsky at University of California Davis for providing EMS mutants of durum wheat Kronos. This work was supported by the National Natural Science Foundation of China (No. 31370304), the National Key Research and Development Program of China (2016YFD0101004) and by Funds of Shandong “Double Tops’’ Program. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.plantsci.2018.10.024. References [1] W.Q. Wang, Q.Q. Hao, F.X. Tian, W. Wang, The stay-green phenotype of wheat mutant tasg1 is associated with altered cytokinin metabolism, Plant Cell Rep. 35 (2016) 585–599. [2] G. Spano, N.D.I. Fonzo, C. Perrotta, Physiological characterization of ‘stay green’ mutants in durum wheat, J. Exp. Bot. 54 (2003) 1415–1420. [3] S. Ha, R. Vankova, K. Yamaguchi-Shinozaki, K. Shinozaki, L.P. Tran, Cytokinins: metabolism and function in plant adaptation to environmental stresses, Trends Plant Sci. 17 (2012) 172–179. [4] J. Van Staden, E.L. Cook, L.D. Noodèn, Cytokinins and senescence, in: L.D. Noodèn, A.C. Leopold (Eds.), Senescence and Aging in Plants, Academic Press, San Diego, 1988, pp. 281–328. [5] E. Taverner, D.S. Letham, J. Wang, E. Cornish, D.A. Willcocks, Influence of ethylene on cytokinin metabolism in relation to Petunia corolla senescence, Phytochemistry. 51 (1999) 341–347. [6] P.R.H. Robson, I.S. Donnison, K. Wang, Leaf senescence is delayed in maize expressing the Agrobacterium IPT gene under the control of a novel maize senescenceenhanced promoter, Plant Biotechnol. J. 2 (2004) 101–112. [7] M.L. Cao, Performance of autoregulatory senescence-inhibition in rice, Hum. Agric. Agric. Sci. Technol. News l2 (2001) 17–24. [8] Y.J. Lin, M.L. Cao, C.G. Xu, H. Chen, J. Wei, Q.F. Zhang, Cultivating rice with delaying leaf senescence by P-SAG12-IPT gene transformation, Acta Bot. Sin. 44 (2002) 1333–1338. [9] C. Masclaux, I. Quillere, A. Gallais, B. Hirel, The challenge of remobilization in plant nitrogen economy. A survey of physio-agronomic and molecular approaches, Ann. Appl. Biol. 138 (2001) 69–81. [10] A.K. Borrell, G.L. Hammer, E. Van Oosterom, Stay-green: a consequence of the balance between supply and demand for nitrogen during grain filling, Ann. Appl. Biol. 138 (2001) 91–95. [11] E.J. Van Oosterom, A.K. Borrell, S.C. Chapman, I.J. Broad, G.L. Hammer, Functional dynamics of the nitrogen balance of sorghum: I. N demand of vegetative plant parts, Field Crop Res. 115 (2010) 19–28. [12] P. Martre, J.R. Porter, P.D. Jamieson, E. Triboi, Modelling grain nitro-gen accumulation and protein composition to understand sink/source regulations of nitrogen remobilization for wheat, Plant Physiol. 133 (2003) 1959–1967. [13] O. Gaju, V. Allard, P. Martre, J.L. Gouis, D. Moreau, M. Bogard, Nitrogen
4.2. cisZOGT1-regulated CK and N content in the delayed senescence of plants Glycosylation plays an important role in plant growth and development; O-glycosylation, is thought to have an effect on the balance of hormones [36]. It is assumed that CK O-glucosides represented reversibly inactivated storage forms [17]. The cisZOGTs preferentially catalyzed the O-glucosylation of cis-zeatin in vitro [37]. Transgenic maize over-expressing the cisZOGT1 gene presented the CK deficient phenotype, although it also showed the delayed senescence phenotype [17]. In this study, the knock-out lines of the cisZOGT1 gene, mutant 2885-1, 2885-2, and 2885-3, exhibited the delayed senescence phenotype at the late filling stage (Fig. 8A). Moreover, the total N, ZR, and 78
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