Participation of ABA Metabolism and ROS Generation in Sugar Starvation-Induced Senescence of Rice Flag Leaves

Background: Both sucrose and abscisic acid (ABA) play pivotal role in the regulation of plant leaf senescence. However, the exact mechanism by which sugar starvation , ABA, and reactive oxygen species (ROS) interact with each other during leaf senescence remains largely unknown. In this study, the genotype-dependent alteration in temporal patterns of sugar concentration during leaf senescence and its relation to ABA metabolism and ROS generation were investigated by using the premature senescence of flag leaf ( psf ) mutant and its wild type. Results: Results showed that sugar starvation-induced leaf senescence was closely associated with the endogenous ABA concentration and ROS level in senescent leaves. Sugar starvation accelerated leaf senescence, concomitantly with the marked increase in ABA concentration and malonaldehyde (MDA) accumulation in detached leaves. Conversely, exogenous sugar treatment significantly suppressed the ABA concentration ad ROS level in detached leaves, thus leaf senescence was delayed by exogenous sugar supply. Pharmacological tests revealed that ABA biosynthesis inhibitor (NDGA) delayed the sugar starvation-induced leaf senescence, while ABA catabolism inhibitor (DNCZ) accelerated leaf senescence and significantly increased the endogenous ABA content in senescent leaves. For the expression patterns of ABA synthesis and catabolism related genes induced by sugar starvation, exogenous sucrose supply, NDGA and DNCZ. sugar starvation upregulated the OsABA8ox1 transcript, while exogenous sucrose and NDGA downregulated the transciptional expressions of OsNCED1 , OsNCED4 and OsNCED5 and OsABA8ox2 and OsABA8ox3 e by sugar starvation and DNCZ, while the transcript of was increased.

alteration in temporal patterns of sugar concentration during leaf senescence and its relation to ABA metabolism and ROS generation were investigated by using the premature senescence of flag leaf ( psf ) mutant and its wild type.
Results: Results showed that sugar starvation-induced leaf senescence was closely  Conclusion: Together, our results demonstrated that the rise in endogenous ABA content during sugar starvation-induced leaf senescence is mostly caused by the suppression of ABA catabolism, rather than the enhancement of ABA biosynthesis, and the expression of ABA metabolic genes determines the equilibrium between ABA biosynthesis and catabolism that eventually influence cross-talk between endogenous factors. The breaking for the equilibrium between ABA biosynthesis and catabolism was strongly responsible for sugar starvation-induced leaf senescence, which was resulted from the suppression of ABA catabolism, rather than the enhancement of ABA biosynthesis .

Background
Leaf senescence is the end of plant developmental phase starts with complex metabolic and physiological changes of chlorophyll (Chl) loss, decreased photosynthetic efficiency, destruction of chloroplast ultra-structures and remobilization of the nutrients, which altogether lead to the death of senescing leaves [1,2]. The initiation of destructive metabolic pathways increase lipid peroxidation and membrane leakage, reactive oxygen species (ROS) generation and plant growth, tolerance to external stresses (sugar homeostasis) and productivity. A little deviation in sugar status (sugar starvation) integrate with plant signaling network of hormones to minimize biosynthesis, cellular respiration and cellular growth [7]. The persistence of sugar starvation for long time regulates catabolic activities, oxidative level of cell and nutrient remobilization from vegetative tissues to rice storage grains. Previous studies indicate that dark induced sugar starvation reduce photosynthetic activity, and induce ubiquitin process of chlorophyll degradation [8][9][10][11]. The dark induced sugar starvation studies reported the up or down regulation of 800 genes related to the cellular catabolism in Arabidopsis cell suspension cultures [12]. At cellular level, sugar starvation activates a catabolic network responsible for breakdown of starch, lipids, proteins, and other cellular constituents which ultimately damages the cell anabolic function. This sugar starvation induced catabolic activities creates redox imbalance/oxidative stress by excessive ROS accumulation in plant cell. Abscisic acid (ABA) is the prime shield among internal regulatory factors against external stresses and works as a link between oxidative damage of cellular structure and sugar signaling [13][14][15]. If the extent of sugar starvation and oxidative stress dominates and persist for long time, then ABA regulate the onset of leaf senescence [6]. However, the exact mechanism by which sugar starvation function ABA accumulation and its interaction with ROS accumulation during senescence remains unclear. The tri-sense loop of sugar starvation, oxidative stress and ABA regulate leaf senescence in complicated manner which is the actual topic of ours debate.
In present study, we used the rice premature senescence of flag leaf (psf) mutant and its wild type to assess the genotypic-dependent alteration in sugar, sucrose, hexose, MDA, ROS and ABA contents and their temporal pattern during leaf 6 senescence. Meanwhile, detached leaf segments were employed to in vitro dark treatment, and exogenous sucrose and mannitol treatment, to investigate the effects of artificial sugar starvation on leaf senescence and senescence-related parameters in rice. Moreover, the relationship between sugar starvation and ABA synthesis and catabolism was studied by applying treatments with ABA anabolism inhibitor nordihydroguaiaretic acid (NDGA) and ABA catabolism inhibitor diniconazole (DNCZ) was performed to clarify the link between sugar starvation and ABA metabolism during leaf senescence.

Results
Genotype-dependent alteration in the senescence-related physiological parameters and their temporal pattern during leaf senescence for the psf mutant and its wild type No visible differences between the psf mutant and the wild type were observed at the seedling and early tillering stages. However, leaf senescence symptoms appeared initially on the lower leaves of the psf mutant at the late tillering stage, and the exacerbated lesions subsequently extended to the upper leaves. The flag leaf of psf mutant exhibited senescence symptoms post anthesis, and the lesions first appeared on the leaf tip and then spread gradually downward to cover the whole leaf blade and the leaf sheath. Subsequently, the flag leaf of psf mutant was completely withered approximately at 30 days post anthesis. By contrast, the leaves in the same position of wild type still remained green during the same period ( Fig.   1). The chlorophyll content in the flag leaves of psf mutant dropped earlier than those of its wild type, with more sharply descending pattern for the psf mutant (Fig.  2B). Meanwhile, the MDA accumulation in psf flag leaves increased rapidly after anthesis, but this trend was not observed in the wild type, which showed stable MDA content or a slight increase in MDA content from 0 to 28 days post anthesis (Fig.   2D). Similar to MDA content, the psf mutant had significantly higher H 2 O 2 concentration in flag leaves than its wild type. Interestingly, the ABA concentrations in the psf leaves increased quickly from 7 days to 28 days post anthesis, whereas those in the wild cultivar increased slightly until rice harvest (Fig. 2I). These results and OsABA8ox3 genes expressed preferentially in rice leaves, with relatively low transcription or undetectable levels in culm and grain (Fig. 5H). OsNCED1, OsABA8ox2 and OsABA8ox3 genes were highly expressed in the leaves and sheaths and moderately expressed in the culm and grains, compared to those in the psf mutant, these genes displayed higher expression in wild type. OsNCED5 gene was only detectable expressed in the leaf, whereas OsNCED2 was only lowly expressed in the culm in the psf mutant and its wild type, and OsABA8ox1 was lowly expressed in various organs, with slight difference between the two genotypes. Thus, OsNCED1 and OsABA8ox3 expressions were more closely related to the alteration of ABA levels in the genotype-dependent senescence than the other ABA metabolic regulatory genes during sugar starvation-induced leaf senescence.
For five OsNCEDs and three OsABA8ox isoforms, OsNCED1 and OsABA8ox3 were highly expressed in rice leaves, and the transcriptional levels of OsNCED4, OsNCED5, OsABA8ox1, and OsABA8ox2, were moderately or lowly abundant for two rice genotypes, whereas the transcripts of OsNCED2 and OsNCED3 were undetectable due to an extremely low level oftranscriptional expression (Fig. 5H).
Comparatively, the psf mutant generally had remarkably lower transcripts of OsNCED1 and OsABA8ox3 than its wild type, with the reducing trend along with leaf senescence ( Fig. 5B and 5G). This result implied that the down-regulation of OsNCED1 and OsABA8ox3 participated in the regulation of ABA concentration in psf mutant leaves, which might be closely associated with the genotype-dependent alteration in the invitation and progression of leaf senescence. Considering the strongly elevated ABA concentration during leaf senescence and the genotypedependent alteration in leaf senescence process, we presumed that the strikingly increased ABA concentration in the senescent leaves of psf mutant was more attributable to the suppression of ABA catabolism, rather than the enhancement of ABA biosynthesis during leaf senescence.
To elucidate the relationship of ABA biosynthesis and catabolism with sugar starvation-induced leaf senescence, we further examined the transcripts of several genes involving in ABA biosynthesis and catabolism by using the detached leaf segments under darkness. The result showed that the transcripts of OsNCED1, OsNCED4, and OsABA8ox3 in psf mutant leaves was more severely suppressed than its wild after 6 day's darkness. OsNCED4 expression in psf mutant leaf was downregulated by approximately 5-fold after 6 day dark treatment, whereas only 0.5-fold decrease was observed for wild type at the same incubation stage (Fig. 6).
By contrast, the transcript of OsABA8ox1 was significantly enhanced by sugar starvation treatment after 3 day and 6 day incubation under darkness (Fig. 6D).
These results further indicated that the increasing levels of ABA in sugar starvationinduced leaf senescence were mainly attributable to the weakening of ABA

Discussion
Sucrose is the main end-product of photosynthesis, used for the transport and redistribution of photosynthetic products in higher plants [16]. The little deviation of sugar due to environmental stresses affect sugar signaling and mediate regulatory effect of sugar on the leaf senescence [8,17]. For instance, the 13 endogenous sugar concentrations markedly decreased during drought induced leaf senescence in sorghum [9]. Our study found the reduced soluble sugar, sucrose and hexose in senescent flag leaves of psf mutant. The darkness treatment of detached leaf segments of the psf mutant and its wild type induce leaf senescence. Contrarily to our findings, One et al. (1996) found that endogenous sugar contents of sunflower increased leaf senescence in low N. In contrast to sugar accumulation hypothesis [18], high sucrose concentration by > 160 mM delayed the onset of leaf senescence in detached leaf segments in our study. These observations are supported by previous findings of Fujiki et al. [19] and Xiao et al. [20] in Arabidopsis. The imbalance of sugar and sucrose ratios in senescent leaves has been accredited in previous studies on sugars starvation-induced leaf senescence [21]. In this study, we found that the decline of hexose was significantly less than that of sucrose in the early stage (from 0 day to 7 DAA) of flag leaf senescence in psf mutant. The sugar starvation treatment of detached leaf segments also exhibited similar trend (Fig. 3H). This early decline of hexose content in the psf leaf (at 0-day post anthesis) may have accelerated the unloading of sucrose from the leaf and its decomposition into hexose, and a result the changing rate of hexose to sucrose was increased. Therefore, the increased fluctuating ratio of hexose and sucrose greatly contribute to the onset and acceleration of sugar starvation induced leaf senescence during the grain filling stage. Low cell-wall invertase activity by INVINH1 gene repression reduces the apoplasmic hexose level that eventually promote the transport of sucrose from leaf carbohydrate storage to phloem, and thus reducing carbohydrate level in leaves which cause leaf senescence.
ABA being the most important stress responsive plant hormone is involved in regulation of many physiological and developmental processes, such as stomatal closure during drought, dormancy of seeds under extreme environmental conditions and leaf senescence due to internal and external factors [6]. Research involving physiological and genetic analysis have affirmed a positive correlation between sugar and ABA signaling [22,23].  were measured by following the procedure as described by Rao et al. [42]. For all these parameters described above, the flag leaves and/or its detached leaf segments were used for each measurement, with 3-4 biological replicates.

21
Extraction and determination of ABA in rice leaves The extraction and purification of ABA in rice flag leaves was carried out according to the method of Kojima et al. [43] with some modifications. One-gram aliquot of fresh leaf samples was homogenized in liquid nitrogen and added to 5 ml of frozen extraction buffer (methanol: formic acid: water = 15: 1: 4). After 24 h extraction at -20 °C, the homogenate was centrifuged at 10,000 × g for 15 min at 4 °C. The supernatants from each sample were transferred to a 96-well collection plate and the remaining residues were extracted again. Subsequently, the collection plate containing the sample was placed in the automatic extraction of solid phase extraction system (SPE215; Gilson, Middleton, WI, USA). After extraction, the residual extraction was activated with 1 mol L -1 formic acid before through column.
The ABA was washed out using methanol. and the eluent was evaporated and further purified. Afterward, ABA content was measured by UPLC-ESI-qMS/MS method.
The chromatographic conditions and MS parameters in UPLC-ESI-qMS/MS system were designed as described by Kojima  Each experiment was performed with three replicates.

Statistical analysis
All determinations were performed in at least three independent experiments.
Statistical differences were analyzed by analysis of variance (ANOVA) using the SPSS statistical software package (Chicago, Illinois, USA). The mean were compared by the least significant difference (LSD) test at probability level of p < 0.05.

Funding
The authors are deeply indebted to National Key Research and Development Plan of China (No 2

Ethic approval and consent to participate
Not applicable

Consent for publication
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Availability of data and materials
All data and materials generated or analyzed during this study are included in this article or are available from the corresponding author on reasonable request