Leaf senescence induced by EGY1 defection was partially restored by glucose in Arabidopsis thaliana

Abstract

Background

Ethylene-dependent gravitropism-deficient and yellow-green 1 (EGY1) protein is required for chloroplast development and photosynthesis conduction. The egy1 deletion mutants have a yellow-green phenotype and reduced granal thylakoids. Furthermore, the yellow-green phenotype of egy1 mutants is more obvious than that of wild-type (WT) plants with increasing leaf age, suggesting an early senescence in the egy1 mutants. However, the relationship between EGY1 functions and leaf senescence still remains poorly understood.

Results

We observed that egy1 mutant leaves were more yellow than those of WT (the same age) in Arabidopsis thaliana. In accompany with this phenotype, leaf survival, chlorophyll content, Fv/Fm and soluble protein content decreased, and ion leakage increased significantly in egy1 mutants compared to WT plants. At molecular level, the expressions of senescence-associated genes increased, and photosynthesis genes decreased significantly in the mutants compared to those in WT plants. Furthermore, after darkness treatment, the yellow-green phenotype of egy1 mutants was more obvious than that of WT. These results indicate that the loss-of-function of egy1 gene induces leaf senescence in A. thaliana. In addition, our results showed that the yellow-green phenotype, chlorophyll content and ion leakage of egy1 mutants was partially restored after exogenously applied glucose for 5 weeks. At the same time, the expression of hexokinase 1 (HXK1) and/or senescence-associated gene 12 (SAG12) in egy1 mutants growing on 2 % glucose was lower than that in egy1 mutants without glucose.

Conclusion

EGY1-defection induced leaf senescence and this senescence was partially restored by glucose in A. thaliana.

Chen et al. Bot Stud (2016) 57:5

DOI 10.1186/s40529-016-0120-3

ORIGINAL ARTICLE

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Web End = Cuiyun Chen*, Jin Wang and Xin Zhao

Abstract

Background: Ethylene-dependent gravitropism-decient and yellow-green 1 (EGY1) protein is required for chloroplast development and photosynthesis conduction. The egy1 deletion mutants have a yellow-green phenotype and reduced granal thylakoids. Furthermore, the yellow-green phenotype of egy1 mutants is more obvious than that of wild-type (WT) plants with increasing leaf age, suggesting an early senescence in the egy1 mutants. However, the relationship between EGY1 functions and leaf senescence still remains poorly understood.

Results: We observed that egy1 mutant leaves were more yellow than those of WT (the same age) in Arabidopsis thaliana. In accompany with this phenotype, leaf survival, chlorophyll content, Fv/Fm and soluble protein content decreased, and ion leakage increased signicantly in egy1 mutants compared to WT plants. At molecular level, the expressions of senescence-associated genes increased, and photosynthesis genes decreased signicantly in the mutants compared to those in WT plants. Furthermore, after darkness treatment, the yellow-green phenotype of egy1 mutants was more obvious than that of WT. These results indicate that the loss-of-function of egy1 gene induces leaf senescence in A. thaliana. In addition, our results showed that the yellow-green phenotype, chlorophyll content and ion leakage of egy1 mutants was partially restored after exogenously applied glucose for 5 weeks. At the same time, the expression of hexokinase 1 (HXK1) and/or senescence-associated gene 12 (SAG12) in egy1 mutants growing on 2 % glucose was lower than that in egy1 mutants without glucose.

Conclusion: EGY1-defection induced leaf senescence and this senescence was partially restored by glucose in A. thaliana.

Keywords: egy1, Leaf senescence, Glucose, Senescence-associated genes

Background

The leaf is a specialized photosynthetic organ of plants, serving as the major site of producing energy and nutrients. Its development stages include initiation, growth, dierentiation, maturation and senescence. Leaf senescence, the last stage of leaf development, plays a key role in plant survival and/or death. During leaf senescence, plant cells undergo orderly changes in structure, metabolism and gene expression (Buchanan-Wollaston et al. 2003; Guiboileau et al. 2010). Among these, the

degradation processes of chlorophyll, proteins and lipids have been largely investigated (Hortensteiner and Feller 2002; Wiedemuth et al. 2005). Chlorophyll degradation typically starts at the leaf margins and progresses to the interior of the leaf blade (Kakani etal. 2004). Protein degradation is the most signicant breakdown process that takes place during senescence (Buchanan-Wollaston etal. 2003). Other organelles, such as the liposome, also undergo biochemical changes as senescence proceeds (Hung and Kao 1998).

Leaf senescence is usually triggered by internal age-dependent factors, which include the expression changes of senescence-associated genes (SAGs), and the mutants of leaf death inducing genes, jasmonic acid-associated

*Correspondence: [email protected] Physiology and Ecology Laboratory, Cold and Arid Regions Environment and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, China

2016 Chen et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/

Web End =http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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genes and other key genes (Castillo and Leon 2008; Guo etal. 2004). However, some external factors such as the alterations of nutrient, light and other environmental factors also initiate leaf senescence (Guiboileau etal. 2010; Ono etal. 2001). Of course, the defect of a gene associated with producing a nutrient (such as sugar) has the same eects as the external factors mentioned above (Loreti etal. 2008). Sugar signaling pathways are important regulation mechanisms for leaf senescence in plants (van Doorn 2008), which consists of glucose, sucrose, trehalose and other hexokinase-independent sugar signaling pathways (Smeekens etal. 2009; Xiao etal. 2000). Among these, the glucose signaling pathway is considered as one of the most important mechanism responsible for senescence and has been extensively studied (Balasubramanian etal. 2008). And hexokinase 1 (HXK1) acts as the direct glucose sensor mediating multiple functions in the glucose repression and glucose promotion of transcription and growth (Cho etal. 2006).

Nuclear mutations that cause albino or pale green phenotypes because of reduced levels of chlorophyll in the chloroplasts have been found frequently in higher plants (Bellaoui and Gruissem 2004; Vinti et al. 2005). These mutants usually have normal leaf anatomies, but typically show defects in chloroplast ultrastructure and composition (Aluru etal. 2007). The mutants of egy1 (which coding for a protein named ethylene-dependent gravitropism-decient and yellow-green 1, EGY1) are such plants of Arabidopsis thaliana because EGY1 protein is required for chloroplast development and photosynthesis conduction (Chen etal. 2005; Guo etal. 2008), and the yellow-green phenotype of egy1 mutants is more obvious than that of wild-type (WT) plants with increasing leaf age. Although the photosynthesis characteristics of egy1 mutants have been studied extensively, the relation between egy1 mutation and leaf senescence is completely unknown. Since there was little attention given to color mutants and leaf senescence (Yoshida etal. 2002), we aim to explore the eects of EGY1-defection on leaf senescence and to further elaborate its potential mechanism in this paper.

Methods

Plant materials andgrowth conditions

The T-DNA insertion line SALK-134931 was obtained from the Arabidopsis Biological Resource Center (ABRC, Ohio State University), and plants homozygous for the egy1 mutation (also known as egy1-2 in Chen etal. 2005) were used for further analysis. Wild-type and the mutant plants were grown under 10-h-light/14-h-dark cycle with a photon ux density of 120molm2s1 at 22C. To ensure synchronized germination, the seeds were sown and then maintained in darkness for 48h at 4C.

For dark treatment, 3-week-old seedlings of WT and egy1 mutant plants were placed in complete darkness for 3days at 22C.

Complementation ofthe egy1 mutants

For complementation of the egy1 mutants, cDNA containing the EGY1 coding region was amplied by PCR with the sense primer 5-GGATCCAATGGGGACTCTC AC-CAG-3 and antisense primer 5-CGAGCTCTCACT AGTGTACATACATGGC-3. The PCR product was cleaved with BamHI and SacI and cloned into the plant expression vector pSN1301 under the control of the cauliower mosaic virus 35S promoter. The construct was transformed into Agrobacterium tumefaciens strain C58 and introduced into egy1 plants by the oral dip method (Clough and Bent 1998). Transformed plants were selected on MS medium (Murashige and Skoog basal liquid medium; Sigma-Aldrich) containing 50 mg mL1

hygromycin. The success of the complementation procedure was conrmed by PCR analysis and chlorophyll contents detection on the resulting plants.

Measurement ofleaf survival, chlorophyll content, photochemical efficiency, soluble protein andion leakage

All these parameters were measured with the fth rosette leaves, which were harvested at specied days counted from the day of leaf emergence. Leaf survival was determined by the mortality curves, which was the percentage of leaves that didnt show full yellow (full yellow leaf was considered as dead) versus all leaves. Chlorophyll contents were quantied as described by Sims and Gamon (2002).

The photochemical efficiency of PSII was deduced from chlorophyll uorescence parameters measured using a portable plant efficiency analyzer (Hansatech Instruments, Morfolk, England). The ratio of maximum variable uorescence to maximum yield of uorescence (Fv/Fm) was used as a measure of the photochemical efficiency of PSII.

Protein was extracted in 10 mM HepesKOH (pH 8.0), 10mM MgCl2, 330mM sorbitol, 2mM PMSF and concentration was determined with a protein assay kit (Bio-Rad, CA, USA) using bovine serum albumin as a standard. Membrane ion leakage was determined by measuring electrolytes leaked using a digital conductivity.

Nucleic acid preparation andanalysis

Total RNA was extracted from 100 mg of fresh tissues using TRIzol reagent (Invitrogen). For the determination of the genes expression, RT-PCR was performed using the following primers: sense (5-TCGCTTTTGCCGCTGCC GTTAACATTAG-3) and antisense (5-AAAGCTAA CACGAGCACCACCGCGAGG-3). To ensure equal

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Results

Isolation ofegy1 mutants

The T-DNA insertion line (SALK-134931, Fig. 1a) (Columbia background) was obtained from the Arabidopsis Biological Resource Center (ABRC) at Ohio State University. The homozygosity of plants was conrmed by PCR amplifying and sequencing. To study the eect of the T-DNA insertion on At5g35220 expression, we analyzed the results of RT-PCR, which showed that this gene was undetectable in the mutant, while it was normal expressed in WT plants (Fig. 1b). The oldest egy1 mutant leaves had the most intense yellow-green phenotype (Fig.1c). Complementation of egy1 mutants was performed to ensure the function of EGY1 (Fig.1c).

Yellowing ofegy1 rosette leaves

The fth rosette leaves emerged on the same day both in the egy1 mutants and WT plants. The yellow-green of young leaves and albino of old leaves in egy1 mutant (Fig. 2a) was not due to dierence in the number of leaves. Yellowing of the egy1 leaves happened rst at the tip of the leaf blades, later spread to the end of leaf blades. The leaves sometimes developed chlorosis along the midrib (Fig.2bf).

amount of RNA in each sample, RT-PCR analysis of actin cDNA was performed using the following primers: sense (5-AACTGGGATGATATGGAGAA-3) and antisense (5-CCTCCAATCCAGACACTGTA-3).

Northern blot analyses were performed essentially as described previously (Sambrook and Russell 2001). The fth leaves were harvested from WT and egy1 plants at 26, 33 and 40days after germination, and RNAs were separated on a 1.0% agarose/formaldehyde gel, then transferred onto Nylon membranes (Amersham Pharmacia Biotech). The membranes were probed with 32P-labelled cDNA probes specic for senescence-associated gene 12 (SAG12), SAG24, endo-xyloglucan transferase/xyloglucan endo-1,4-beta-d-glucanase (SEN4), chlorophyll a/b-binding protein (CAB), and the rubisco small subunit gene (RBCS). Ethidium bromide staining was used as a loading control. Following high-stringency hybridization and washing, all the blots were exposed to X-ray lm.

Exogenous glucose treatment

Arabidopsis thaliana seeds were sown on half-strength MS plates containing various concentrations of glucose, and plates were placed at 4C for 2days for vernalization. They were germinated and maintained at 22C in darkness for 6days and illuminated at 22C for 12h. The hypocotyl lengths of seedlings were measured.

At the same time, WT and egy1 plants were grown on 2% glucose+MS medium in a 500 cubic cm glass jar for 5weeks, and then the phenotype were observed, the chlorophyll contents, ion leakage were determined, and the expressions of hexokinase 1 (HXK1) and senescence-associated gene 12 (SAG12) were quantied.

Quantitative PCR Analysis

Primer pairs for the quantitative PCR were: 5-GCAGA CTTCTCTGTCCTCTGG-TAG-3 (forward) and 5-TC CAACAACATCTTGTCCAACTGC-3 (reverse) for HXK1; 5-AAGGAGGAAAACAATCGCTAC-3 (forward) and 5-GCAAACTGA-TTTACCGCAAG-3 (reverse) for SAG12; 5-CGTACAACCGGTATTGTGC T-GG-3 (forward) and 5-CTCTCTCTGTAAGGATCT TCATG-3 (reverse) for actin. The quantitative PCR was performed with a Mx3000P Real-Time PCR System (Stratagene, Agilent, USA) using SYBR Green SuperMix (Takara) with the following conditions: 30s at 95C; 40 cycles of 10s at 95C, 10s at 55C and 12s at 72C, with nal melting for 15s at 65C. Melting curve analysis was performed to conrm the specicity of the amplication and to identify putative unspecic products. Actin mRNA, set to 100%, was used as an internal standard in all experiments. The quantitative PCR experiments were repeated at least three times for a cDNA prepared from three batches of plants.

Fig. 1 Expression of EGY1 and phenotypes of egy1 mutants. Gene structure of EGY1 and location of T-DNA insert (a), RT-PCR analysis of EGY1 expression in wild-type and egy1 leaves (b), phenotypes of wild-type, egy1 and egy1 complemented lines at 28 days after germination (c)

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Fig. 2 Phenotypes of egy1 mutants in age-dependent senescence.

A photograph of wild-type and egy1 plants (a), visible senescence phenotypes in the egy1 leaves (bf), leaves were numbered from bottom to top. Chlorosis often initiates from midrib (c), and sometimes chlorotic lesions are formed (d) in egy1 leaves. Chlorotic area expands from tip to base on an egy1 leaf (e, f)

Physiological changes inegy1 leaves

In order to examine the physiological state of the egy1 mutants, the survival, chlorophyll content, Fv/Fm, soluble protein content and ion leakage of the fth leaves were measured in this study.

We rst examined the eect of visual leaf longevity (representing the leaf survival) in the egy1 mutation (Fig.3a). We observed that the leaf longevity of egy1 was shortened by 40.1%. The time it took 50% of leaf population to survive was 22days after leaf emergence in the mutants, while it was longer, 37days, in the WT plants (Fig.3a).

The chlorophyll content of egy1 mutants at 10 days of leaf emergence was <50 % of that in the WT plants (Fig.3b). Chlorophyll content decreased in egy1 mutants after 20 days, while it was stable until 30 days of leaf emergence in WT leaves (Fig.3b).

The Fv/Fm ratio, which reects the photochemical quantum efficiency of PSII, was similar in egy1 mutants and WT plants at 15 days of leaf emergence. But after 20days, the Fv/Fm ratio began to decrease in egy1 leaves, whereas it did not decline until 25 days in WT leaves (Fig.3c).

At 10 days after leaf emergence, soluble protein contents were measured in egy1 mutants and WT plants. Soluble protein began to decrease on the 15 days of leaf emergence in egy1 mutant, while it increased until 20days in WT leaves. Protein contents decreased below 60 % of the initial level at 25 days of leaf emergence in egy1 mutants and after 35days in WT (Fig.3d).

Ion leakage, an indicator for the intactness of the plasma membrane, began to increase 15 days after leaf emergence in egy1 leaves, and its ratio continued increasing signicantly throughout the growth period. However, ion leakage in WT leaves began to increase until 30days. Moreover, there was no increase in the ratio throughout the growth period in WT plants, unlike egy1 mutants (Fig.3e).

Transcription ofsenescencerelated genes inthe egy1 mutants

To evaluate leaf senescence in the egy1 mutants at molecular level, the transcription levels of SAG12, SAG24, SEN4, CAB and RBCS were examined by RNA gel-blot analysis. The transcripts of SAG12 in egy1 plants accumulated ca. 207 % at 33 days and ca. 400 % at 40 days compared with that of 26days after germination, which was more than those in WT plants (Fig. 4). The transcripts of SAG24 and SEN4 were also increased in egy1 mutants. Similarly, the transcript levels of CAB and RBCS decreased more obviously in egy1 plants than those in WT plants with increasing leaf age (Fig.4).

The inuence ofdarkness treatment

27-Day-old WT and egy1 plants (Fig.5a, b) were placed in a dark chamber in order to observe phenotypic changes due to darkness. After 3days, the yellow-green phenotype was more obvious in egy1 mutants (Fig. 5d) than that in WT plants (Fig.5c).

Leaf senescence ofegy1 mutants was delayed byexogenously applied glucose

Cotyledon greening in WT was almost the same as in egy1 mutants regardless of treatment with or without 1% (56mM) and 2% (111mM) glucose (Fig.6a). However, cotyledons greening was inhibited in egy1 plants treated with 4 % (222 mM) and 6 % (333 mM) glucose, and in WT plants with 6% glucose (Fig.6a). Moreover, hypocotyl elongation was increased from 0 to 2% glucose both in WT and egy1 seedlings (Fig.6b), while it was inhibited under 4% and 6% glucose both in egy1 and WT seedlings (Fig.6b). Next we planted WT and egy1 mutants on 2% glucose for 5weeks. The results showed that yellow-green phenotype of egy1 mutants was partially restored by glucose treatment (Fig.7a). Chlorophyll contents were increased and ion leakage was decreased in egy1 mutants applied with 2% glucose compared to those without glucose treatment (Fig.7b, c).

The results from quantitative PCR analysis indicated that the high expressions of HXK1 and SAG12 in egy1 mutants were impaired by 2 % glucose treatment for 5weeks (Fig.8).

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Fig. 3 A time-course of changes in senescence indicators of the fth leaves at indicated times after leaf emergence. Leaf survival (a), chlorophyll contents per leaf area (b), Fv/Fm (c), soluble protein contents per leaf (d) and ion leakage (e). Soluble protein contents were shown as relative values of those measured at 10 days. At 35 days, chlorophyll contents and soluble protein contents were undetected in the egy1 mutants. The standard deviation was calculated from twelve samples for leaf survival; six for chlorophyll and Fv/Fm, and three for soluble protein and ion leakage measurements

Discussion

In general, leaf senescence is dependent on age and developmental stage under normal conditions and in the absence of external stress, and the leaf yellowing during senescence is usually observed in old leaves. In this study, the leaf yellowing phenotype of egy1 mutant and WT plants is also related to the increasing leaf age and

leaf normal development. However, egy1 mutant leaves are more yellow than those of WT plants at the same age and development stage (Figs.1, 2), suggesting that egy1 mutants have an early-senescence phenotype.

To further conrm the early-senescence phenotype, we measured the physiological characteristics associated with senescence in egy1 mutants. The leaf longevity was

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Fig. 4 Transcript accumulation of senescence-related genes and genes related to photosynthesis in naturally senescing egy1 leaves. The percentages of RNA levels shown above the lanes were estimated by comparison with levels found in wild-type corresponding samples taken at 26 days

Fig. 5 Yellowing of egy1 leaves during dark treatment. Wild-type (a, c) and egy1 (b, d) plants before dark treatment (a, b) and after 3 days (c, d) of dark treatment are shown

Fig. 6 Sugar response in the egy1 seedlings. Wild-type and egy1 seedlings were grown on a medium containing various concentrations of glucose for 6 days under darkness and then for 12 h under illumination. Phenotypes of the seedlings (a), hypocotyl length of seedlings was measured and standard deviations were calculated from 12 seedlings (b)

lower in egy1 mutants than that of WT plants (Fig. 3). Similar results were detected in apg7-1 mutants of Arabidopsis which showed an early-senescence phenotype (Doelling et al. 2002). Chlorophyll can provide basic information of photosynthesis and photosynthesis defects is an important feature of senescence. Our results showed that chlorophyll contents were also reduced in egy1 mutants (Fig.3), which was similar to the reported data by Frick etal. (2003) in porB-1porC-1 double mutant of older seedlings and by Stettler et al. (2009) in mex1 leaves. Proteins are fundamental components of all living cellsand are necessary for the proper functioning of an organism, and could be decreased during leaf senescence. We found that egy1 mutation had decreased soluble protein contents (Fig.3), similar tendency was also detected in hys1 and apg7-1 early-senescence mutants (Doelling etal. 2002; Yoshida etal. 2002). Ion leakage is an indicator of membrane integrity. When leaf senescence, membrane became fragile and leak. In egy1 mutants, ion leakage was

increased (Fig.3). Taken together, these results suggested that the egy1 mutants had early-senescence traits.

Leaf senescence is accompanied by increased expression of senescence-associated genes (SAGs) and decreased expression of genes related to photosynthesis (Lim etal. 2003; Zentgraf etal. 2004). The transcript levels of SAG12, SAG24 and SEN4 genes were increased in senes-cent leaves (Wu et al. 2008; Yoshida et al. 2001), while CAB and RBCS genes were down-regulated during leaf

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Fig. 7 Phenotypes (a), chlorophyll contents (b) and ion leakage (c) in wild-type and egy1 plants supplied with 2 % glucose for 5 weeks

Fig. 8 The relative transcription levels of hexokinase 1 (HXK1) (a) and senescence-associated gene 12 (SAG12) (b) in egy1 and wild-type plants growing on 0 % and 2 % glucose. The transcripts were determined by real-time PCR and normalized against actin. The transcription levels are relative to the WT set at 0 % glucose. The quantitative PCR experiments were repeated at least three times for a cDNA prepared from three batches of plants

senescence (Woo etal. 2001). Similar results were found both in egy1 mutants and in WT plants with increasing leaf age. It is notable that, SAG12, SAG24, SEN4 increased and CAB and RBCS decreased more obviously in egy1 mutants compared to WT plants (Fig.4). These

results indicated that leaf senescence occurred indeed in transcript level in egy1 plants.

Leaf senescence could be induced by external stresses, such as darkness. Darkness treatment induced uniform and rapid senescence (Chrost et al. 2004; del Rio et al.

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Abbreviations

EGY1: ethylene-dependent gravitropism-decient and yellow-green 1; SAG: senescence-associated gene; CAB: chlorophyll a/b-binding protein ; RBCS: the rubisco small subunit gene; SEN4: endo-xyloglucan transferase/xyloglucan endo-1,4-beta-D-glucanase.

Authors contributions

CYC conceived and designed the experiments. CYC XZ performed the experiments. JW analyzed the data. CYC wrote the paper. All authors read and approved the nal manuscript.

Acknowledgements

This research was supported by National Natural Science Foundation of China (31300226), the National Basic Research Program of China (2013CB429904) and the Natural Science Foundation of Gansu Province (1308RJYA094).

Competing interests

The authors declare that they have no competing interests.

Received: 30 June 2015 Accepted: 20 January 2016

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2003; Mishev et al. 2009). After 3 days of dark treatment, egy1 mutant leaves were more yellow while the phenotype of WT plants had no obvious changes (Fig.5), suggesting that egy1 mutants had an early-senescence phenotype under normal conditions and in darkness treatment. So EGY1 should have an important role in leaf normal development in A. thaliana. As the egy1 mutants have a wide variety of senescence symptoms, the egy1 gene most likely functions as a regulatory component controlling functional leaf senescence rather than as a protein directly executing the senescence process (Kim etal. 2009; Lim etal. 2010).

The chloroplasts in egy1 mutants had reduced granal thylakoids, poorly developed lamellae networks and decreased photosynthetic proteins (Chen et al. 2005). The loss in photosynthesis caused cellular sugar starvation, and lack of energy (Patro etal. 2014). Sugar starvation is known to aect leaf senescence, repress hypocotyl elongation, cotyledon greening and shoot development (Arru et al. 2008; Gazzarrini and McCourt 2001). In tobacco plants, sugar starvation accelerated leaf senescence, suggesting that sugar signaling could directly or indirectly promote leaf senescence (Wingler and Roitsch 2008). This starvation could be restored after exogenously applied sugars through sugar metabolism with complex sugar signaling network. Yoshida et al. (2002) found that hys1 seedlings with exogenously applied sugars were hyper-responsive to glucose, implying that sugar may play a role in the early induction of senescence. In our study, hypocotyl elongation was increased in egy1 seedlings growing on 2 % glucose. Furthermore, the yellow-green phenotype of egy1 mutants was partially restored by 2% glucose (Fig.7), suggesting that the symptom of early senescence was relieved after glucose supplementation. Meanwhile, the expressions of HXK1 and SAG12 (which are usually at a high level in senescence leaves) were obviously decreased in egy1 mutants by 2 % glucose (Fig. 8), implying that the early senescence of egy1 mutants may be due to sugar starvation. It therefore could be restored by exogenous glucose treatment. With regard to the inhibition of cotyledons greening or hypocotyl elongation by 4% or 6% glucose, it is a result from the eects of high concentrations of glucose in the medium and has been reported in the previous study (Jang etal. 1997).

Conclusion

In conclusion, our results suggest that leaf senescence induced by EGY1-defection may be due to sugar starvation and can be partially restored by glucose in A. thaliana. EGY1 regulated leaf senescence provides key information to understand the molecular mechanism of leave senescence in plants.

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