Expression of CsSEF1 gene encoding putative CCCH zinc finger protein is induced by defoliation and prolonged darkness in cucumber fruit
- 391 Downloads
To find a marker gene for photoassimilate limitation in cucumber fruit, genes induced in young fruit by total defoliation were cloned using the subtraction method. Almost every clone matched perfectly to a member of cucumber unigene ver. 3 of the Cucurbit Genomics Database. From the clones obtained, six genes were selected and the effect of defoliation on their expression was analyzed. In particular, expression of a gene that is highly homologous to the cucumber gene CsSEF1 (CAI30889) encoding putative CCCH zinc finger protein, which is reported to be induced at somatic embryogenesis in suspension culture, was enhanced by the treatment by about 50 times. The sequencing of the full-length cDNA and BLAST search in the Cucurbit Genomics Database indicated that our cloned gene is identical to CsSEF1. In control fruit, the expression of CsSEF1 did not change markedly in terms of development. By contrast, the expression of CsSEF1 was enhanced by prolonged darkness at the transcript level. This increase in the expression of CsSEF1 was temporally correlated with the decline in the fruit respiration rate. In mature leaves under prolonged darkness, enhanced expression was observed in the asparagine synthetase gene, but not in CsSEF1. These results suggest that the asparagine synthetase gene can be a good marker for sugar starvation and that CsSEF1 might be involved in the signal transduction pathway from photoassimilate limitation to growth cessation in cucumber fruit.
KeywordsCsSEF1 Cucumber Fruit growth Photoassimilate Respiration Tandem CCCH zinc finger
Days after anthesis
Quantitative reverse transcriptase–polymerase chain reaction
Rapid amplification of cDNA ends
Tandem CCCH zinc finger
In cucumber, the production of malformed fruit should be avoided. Although it has long been assumed that the production of malformed fruit is related to limited photoassimilate supply (Kato and Oda 1977), this has not been strictly proven. The regulatory mechanism of photoassimilate partitioning to sink organs is poorly understood (Gifford and Evans 1981; Giaquinta 1983; Thorne 1985; Ho 1988; Frommer and Ninnemann 1995; Lalonde et al. 2004; Marcelis et al. 2004; Turgeon and Wolf 2009; Wubs et al. 2009; Zhou et al. 2009; Nunes-Nesi et al. 2010; Ruan et al. 2010; Chen and Thelen 2011). One possible strategy for studying the effect of limited photoassimilate supply at the cellular level may be to find a marker gene that responds to such a limitation in the fruit. Extensive studies on the effect of sugar status in Arabidopsis transcriptomes have been performed (Price et al. 2004; Blasing et al. 2005). Whereas interest in gene expression induced by sugar or sugar starvation has increased (Rolland et al. 2006), only a few published studies have investigated the relationship between photoassimilate supply and gene expression in rapidly growing fruits such as cucurbits (Craft and Lorentz 1944). Defoliation (Tamura et al. 2011) and prolonged darkness (Baena-González et al. 2007) have been widely used as methods to limit photoassimilate supply. Here, we used complete defoliation as a treatment and the subtraction method to clone genes whose expression is enhanced in young fruit by defoliation. We obtained the full-length cDNA from a clone whose expression was markedly enhanced by defoliation, and identified it as the CsSEF1 gene, which is reported to be induced during somatic embryogenesis in cucumber (Grabowska et al. 2009). The response of CsSEF1 expression differed from that of the asparagine synthetase gene, which is known to be a good marker of sugar starvation. The decline in the fruit respiration rate, which is almost proportional to the fruit growth rate (Tazuke and Sakiyama 1991), temporally coincided with the marked enhancement of CsSEF1 expression. Together, these results suggest that CsSEF1 gene action is located somewhere in the signal transduction pathway from photoassimilate starvation to the growth cessation of sink tissues, but is independent of the local sugar-sensing pathway.
Materials and methods
Cucumber (Cucumis sativus L.) cv. Tokiwa (supplied by Dr. Yoshiteru Sakata of the Institute of Vegetable and Tea Science, National Agriculture and Food Research Organization (NARO), Kusawa, Ano, Tsu, Mie, Japan) was used. The plants for subtraction cloning were grown in autumn 2009. Plants were grown in a glasshouse in 15-L pots filled with vermiculite and irrigated with half-strength Hoagland No. 2 solution. The plants were pinched, leaving 12 leaves. Fruits on the primary node of the lateral shoots were used. Female flowers were pollinated with pollen from male flowers of the same plant in the morning of the day of anthesis. Seven days after anthesis (DAA) at 11 a.m., when fruits were about 9 cm long, a control fruit was harvested. At the same time, the plant was completely defoliated for the fruit treatment. At 11 a.m. the next day, the treated fruit was harvested. The whole fruit (about 5 g) was frozen with liquid N2 and powdered with a mortar and pestle. Total RNA was extracted with ISOGEN (Nippon Gene, Tokyo, Japan). Messenger RNA was purified with the Oligotex™ −dT 30 <Super> mRNA Purification Kit (TaKaRa, Ohtsu, Japan). The extract was concentrated as follows. To the 600-μL extract, 600 μL isopropanol and 120 μL 4 M LiCl were added; the mix was incubated at −20 °C for 10 min and centrifuged at 12,000×g. Water (20 μL) was then added to the pellet, which was incubated at 55 °C. The extracted total mRNA was used for subtraction cloning using the PCR-Select cDNA Subtraction Kit (Clontech, Mountain View, CA, USA). Double-stranded cDNA was synthesized from the mRNA and digested with RsaI. To the two aliquots of the treatment digest (tester), different oligonucleotides (adaptor) were ligated. Each ligated tester was hybridized with the control digest (driver), combined, hybridized with the driver again, and then amplified by polymerase chain reaction (PCR) using a thermal cycler (Dice mini, TaKaRa) using primers specific to the adaptors. Thus, fragments of cDNAs specific to the treatment were amplified. The PCR product was subcloned with a TA vector (pGEM-T Easy, Promega, Tokyo, Japan). About 100 colonies (white and pale blue) were subcultured overnight, centrifuged, and stored at −80 °C. Plasmids were extracted from 78 samples and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies Japan, Tokyo, Japan) as the reagent and the Applied Biosystems 3130xl Genetic Analyzer (Life Technologies). Matches to unigenes (cucumber ver. 3) of the Cucurbit Genomics Database (http://www.icugi.org/) were examined.
For the sequencing of full-length cDNA, plants were grown similarly in autumn 2011. Four DAA, three plants were completely defoliated at 11 a.m. Their fruits were harvested at 11 a.m. the next day, and portions of fruit tissues in the proximal and distal halves were frozen with liquid N2 and powdered with a mortar and pestle. One hundred milligrams of the aliquot was taken and the total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). 5′-Rapid amplification of cDNA ends (RACE) was conducted using the 5′-Full RACE Core Set (TaKaRa) with LA Taq (TaKaRa). The primers were designed from the known sequence obtained above. The PCR product was subcloned and sequenced as above.
Plants for the expression analysis were grown similarly in autumn 2011 and spring 2012. When fruit weight was about 5 g, three control fruits were harvested at 11 a.m. Plants selected for fruit treatment were completely defoliated at the same time. At 11 a.m. the next day, three treated fruits were harvested. Extraction of total RNA was conducted as above using the RNeasy Plant Mini Kit (Qiagen). First-strand cDNA was synthesized from the total RNA extract using the PrimeScript RT Reagent Kit (TaKaRa) and stored at −20 °C. Expression of the six selected genes was analyzed by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) using SYBR Premix Ex Taq™ II as reagent and the Thermal Cycler Dice Real Time System (TaKaRa). For the expression analysis in prolonged darkness, plants were placed in a darkened growth chamber at 25 °C at 10 a.m. when fruit weight was about 5 g. Three fruits were harvested, at 10 a.m. (control) and 4 p.m. that day, and at 1 p.m. the next day. The effect of prolonged darkness on the expression of the two selected genes was examined similarly. For the analysis of the effect of prolonged darkness on the expression of the two selected genes in plant parts other than fruit, plants were placed in a darkened growth chamber at 25 °C at 10 a.m. Mature leaves, the apical region of lateral shoots, and roots were harvested at 1 p.m. the next day. Plants grown hydroponically in Hoagland No. 2 solution were used to sample roots. Expression of the two selected genes was analyzed as above. To analyze developmental change in the expression of the two selected genes in control fruit, three fruits were harvested, at 10 a.m. that day (5 DAA), the next day (6 DAA), and 2 days later (7 DAA), when fruit weight was about 5 g. Expression of the two selected genes was analyzed as above.
A fruit weighing about 5 g still on the vine was put into a double cylindrical fruit chamber (outer diameter 15 cm, inner diameter 5 cm, length 30 cm) that was wrapped in urethane foam and aluminum foil for heat insulation and light shielding to analyze the fruit respiration rate. Openings in the cylinder were sealed with silicon stoppers. To avoid air leak, the fruit peduncle was sealed with soft rubber. The cylinder temperature was maintained at 25 °C by circulating water controlled by a thermostatic machine (ZL-100, Taitec, Koshigaya, Japan). Room air was drawn by a compressor (Hitachi, Tokyo, Japan) and passed through a column of soda lime. The flow rate was maintained at 2.0 L min−1 using a mass flow controller (SEC-E40, Horiba, Kyoto, Japan), and brought into the inner cylinder, where 0.5 L min−1 of the air flow from the opposite opening was drawn and its CO2 concentration was measured with an infrared gas analyzer (VA-3000, Horiba). Analyzer readings were recorded every minute by a data logger (CR10X, Campbell Scientific, Logan, UT, USA). For the defoliation treatment, plants were supplied with light from 6 a.m. to 6 p.m. by two high pressure sodium lamps (IAN-361FL, GS Yuasa, Kyoto, Japan) with a photosynthetic photon flux density 1,400 μ mol m−2 s−1. After a fruit was placed in the fruit chamber, the plant was defoliated completely at 1 p.m. For the prolonged dark treatment, no light was supplied after a fruit was placed in the fruit chamber. The respiration rate was monitored several times.
For the analysis of sugar and starch, harvested tissue was frozen by liquid N2, powdered with a mortar and pestle, and homogenized with 80 % ethanol. The homogenate was extracted by incubating for 15 min at 80 °C, filtrated through a glass filter (GF/F, Whatman, Kent, UK), and made up to 100 mL. The residue was dried at 55 °C for 1 week. The starch content of the ethanol-insoluble residue was analyzed with F-kit (Roche Diagnostics, Basel, Switzerland). Aliquots of the ethanol extract were dried with a rotary evaporator and dissolved in water. Concentrations of glucose, fructose, and sucrose, which are the major sugars in cucumber fruit (Pharr et al. 1977), were then determined by F-kit. Hereafter, the sum of glucose and fructose concentrations is referred to as the hexose concentration.
Statistical analyses of the significance of treatment effects on the transcript level were conducted using R (R Development Core Team 2011).
Clones obtained by subtraction cloning
After defoliation, fruit grew slightly for about 1 day and increased in volume by about 40 %. They then stopped growing, but stayed green without signs of withering. Fruit harvested a few days after defoliation remained fleshy.
Description of clones obtained
Most closely matched homolog in SwissProt
Number of clones
Probable aquaporin TIP2-2
Zinc finger CCCH domain-containing protein 20
Chlorophyll a/b-binding protein of LHCII type 1, chloroplastic
Phenazine biosynthesis-like domain-containing protein 2
GDSL esterase/lipase 5
Tublin alpha-3/alpha-5 chain
ATP synthase gamma chain, chloroplastic
Photosystem II reaction center W protein, chloroplastic
Chlorophyll a/b-binding protein 3C, chloroplastic
5′-RACE of clone No. 14
Expression of the six selected unigenes after defoliation treatment
As subtraction cloning can yield false-positive results, expression of the cloned genes was examined. Because of the sample limitations, six unigenes were selected for expression analysis. The four most abundant unigenes were included and the remaining two were selected arbitrarily, CU092580 and CU100456, the latter of which was homologous to one clone that is highly homologous to the chlorophyll a/b-binding protein of LHCII type 1 (chloroplastic). The BLAST search of cucumber genome ver. 2 revealed two genes highly homologous to the asparagine synthetase gene. The primers for qRT-PCR were designed to be specific to unigene CU114757.
Detailed expression analysis of CsSEF1 and the asparagine synthetase gene
Carbohydrate concentration of leaves and fruits
It has been suggested that water and minerals enter fruit mainly via phloem (Ho et al. 1987) although this view was recently criticized based on a nuclear magnetic resonance imaging study (Windt et al. 2009). Xylem flow is mainly driven by leaf transpiration (Nobel 2009). These facts suggest that defoliation treatment must have a drastic influence on both phloem and xylem flows, and it is possible that the effects of defoliation can be caused by water and/or ion stresses rather than by photoassimilate starvation. To rule out the possibility that water and/or ion stresses caused the effects of defoliation, prolonged darkness treatments were conducted. Although this kind of treatment also affects phloem and xylem flows, it is much milder than defoliation because no severing of these conduits occurs.
As seen, the transcript level of CsSEF1 increased markedly after prolonged darkness. This strongly suggests that the initial cause of the upregulation of CsSEF1 by defoliation is photoassimilate starvation. Grabowska et al. (2009) reported that the expression of CsSEF1 is correlated with cucumber somatic embryogenesis. To ensure fruit set, we used pollinated fruit as the material. Because the fruits treated with defoliation and prolonged darkness were 1 day older than the control fruits, the increase in the transcript level of CsSEF1 might have been due to developmental changes in the embryo. However, the transcript level of CsSEF1 in the control fruits was very low compared with that in the treated fruits, irrespective of developmental stage, which indicates that the enhancement effect was due to the defoliation or prolonged darkness treatment.
An aspect requiring further examination is whether the site of marked CsSEF1 induction in fruit tissue is concentrated in the embryo. In this regard, it is noteworthy that enhanced expression of CsSEF1 was observed under prolonged darkness not only in fruit, but also in the apical region of lateral shoots and in the roots. However, it was not observed in mature leaves. This suggests that CsSEF1 expression might be confined to rapidly growing sink tissue. It is unclear how to reconcile this view with the induction of CsSEF1 during somatic embryogenesis.
The observed decline in leaf starch concentration is consistent with photoassimilate depletion, but the absence of a difference between 6 and 27 h in darkness and the lack of a marked decline in hexose concentration are in contrast to the response of CsSEF1 expression and the fruit respiration rate. The relationship between fruit abortion and sugar concentration is controversial (Reed and Singletary 1989; Zinselmeier et al. 1999; Marcelis et al. 2004). Despite years of research, the control mechanism for photoassimilate partitioning remains poorly understood (Gifford and Evans 1981; Giaquinta 1983; Thorne 1985; Ho 1988; Frommer and Ninnemann 1995; Lalonde et al. 2004; Marcelis et al. 2004; Turgeon and Wolf 2009; Wubs et al. 2009; Zhou et al. 2009; Nunes-Nesi et al. 2010; Ruan et al. 2010; Chen and Thelen 2011). It is possible that CsSEF1 is related to the cessation of sink growth, i.e., the reduction in sink activity. Examination of CsSEF1 function in the signal transduction pathway could provide some insight into the control mechanism of photoassimilate partitioning.
Arabidopsis and rice genomes have 68 and 67 CCCH zinc finger genes, respectively (Wang et al. 2008). CCCH proteins are found in a range of organisms, from humans to yeast (Nie et al. 1995; Mello et al. 1996; Thompson et al. 1996; De et al. 1999), and are suggested to be RNA-binding proteins functioning in RNA processing (Lai et al. 2000, 2003). CCCH proteins in plants are poorly characterized compared with those in animals. In Arabidopsis, a CCCH zinc finger gene, HUA1, is reported to participate in the regulation of flower development (Li et al. 2001). Another CCCH zinc finger gene, PE11, is involved in embryogenesis (Li and Thomas 1998). Other reported plant CCCHs include ArabidopsisAtCPSF30 (Delaney et al. 2006), FES1 (Schmitz et al. 2005), and rice OsDOS (Kong et al. 2006). Specifically, CsSEF1 has a tandem CCCH zinc finger (TZF) motif. In animals, TZF proteins are characterized by two identical Cx8Cx5Cx3H motifs separated by 18 amino acids (Blackshear et al. 2005). Each CCCH zinc finger is capable of binding to specific RNA motifs (Carrick et al. 2004; Hudson et al. 2004; Barreau et al. 2005). In humans, the TZF family consists of three genes: TTP, BRF1, and BRF2. The functions of these TZFs, especially TTP, have been well studied, and include mRNA degradation (Carballo et al. 1998; Lai et al. 2006). Arabidopsis, rice, and soybean have 11, 9, and 23 TZF genes, respectively (Pomeranz et al. 2011). However, the function of TZF genes in plants is poorly understood, compared with that in animals (Pomeranz et al. 2011). In plants, TZF genes with a plant-specific motif of Cx7-8Cx5Cx3H-x16-Cx5Cx4Cx3H are found. The CsSEF1 gene has this motif. Unlike other plant CCCH families, there has been no report on the specific RNA that binds to plant TZFs (Pomeranz et al. 2011). In Arabidopsis, a TZF gene, AtTZ1 (AtC3H23), was identified in a transcriptome analysis as a glucose-responsive gene (Price et al. 2004). AtTZ1 binds both DNA and RNA in vitro, and traffics between the nucleus and cytoplasm (Pomeranz et al. 2010). Lin et al. (2011) indicated that the expression of AtTZ1 is reduced by glucose in a hexokinase-dependent manner and suggested that AtTZF1 serves as a regulator connecting sugar, abscisic acid, gibberellic acid (GA), and peptide hormone responses.
Thus, the response of CsSEF1 in the present study seems to be different from that of AtTZF1. As shown in Fig. 3, AtTZF1 (AtC3H23) is moderately close to CsSEF1. It will be interesting to see if the expression of more closely related homologs such as AtC3H49 and AtC3H20 is enhanced by prolonged darkness. If so, it will be interesting to see if such enhancement is influenced in hexokinase knockout plants. Lee et al. (2012) studied AtC3H49 and AtC3H20 in relation to GA and jasmonic acid responses.
In conclusion, our results confirm that the asparagine synthetase gene is a good marker gene of sugar starvation in cucumber fruit. The CsSEF1 gene may be involved in the signal transduction pathway that leads to growth cessation of sink organs, but dependence of CsSEF1 expression enhancement on the hexokinase pathway should be further examined.
We thank Dr. Yoshiteru Sakata of the Institute of Vegetable and Tea Science, National Agriculture and Food Research Organization, for kindly offering the seeds of the cucumber cultivar ‘Tokiwa’. This work was supported by Grant-in-Aid for Scientific Research (C) (22580285) on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan to TA.
- Ho LC, Grange RI, Picken AJ (1987) An analysis of the accumulation of water and dry matter in tomato fruit. Plant Cell Environ 10:157–162Google Scholar
- Kato T, Oda H (1977) Studies on the control of physiological disorders in fruit vegetable crops under plastic films. VIII. On the occurrence of abnormal fruits in cucumber plants. (II) On the development of carrot type and bottle gourd type fruits, so-called sakibosori and shiributo fruits in Japan. (Japanese text with English abstract) Res Rep Kochi Univ 26 (Agric Sci):175–182Google Scholar
- Nobel PS (2009) Physicochemical and environmental plant physiology. Academic Press, London, pp 439–505Google Scholar
- Pharr DM, Sox HN, Smart EL, Lower RL (1977) Identification and distribution of soluble saccharides in pickling cucumber plants and their fate in fermentation. J Amer Soc Hort Sci 102:406–409Google Scholar
- R Development Core Team (2011) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/
- Tazuke A, Sakiyama R (1991) Relationships between growth in volume and respiration of cucumber fruit attached on the vine. J Japan Soc Hort Sci 59:745–750Google Scholar