JXB Advance Access originally published online on March 29, 2005
Journal of Experimental Botany 2005 56(415):1397-1407; doi:10.1093/jxb/eri141
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Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.
RESEARCH PAPER |
Regulation of flooding tolerance of SAG12:ipt Arabidopsis plants by cytokinin
1Department of Horticulture and Crop Science, Ohio State University, Columbus, OH 43210, USA
2USDA-ARS-Soil Drainage Research Units, Columbus, OH 43210, USA
3USDA-ARS National Forage Seed Production Research Center, Corvallis, OR 97331, USA
* To whom correspondence should be addressed. Fax: +1 301 838 0208. E-mail: tvantoai{at}tigr.org
Received 2 November 2004; Accepted 18 February 2005
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Abstract |
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A SAG12:ipt gene construct, which increases cytokinin biosynthesis in response to senescence, was introduced into Arabidopsis plants to delay senescence induced by flooding stress. Two forms of flooding stress, including total submergence and root waterlogging, were applied to SAG12:ipt (IPT) and wild-type (WT) plants for 1, 3, and 5 d. A separate experiment compared the recovery of WT and IPT plants subjected to flooding stress. Biomass accumulation, carbohydrate and chlorophyll contents, and cytokinin and abscisic acid were quantified to compare genotypic responses to flooding stress and post-flooding recovery. Real-time RT-PCR studies were performed to quantify ipt and SAG12 gene expression. IPT plants exposed to waterlogging accumulated greater quantities of cytokinins more rapidly than WT plants or those exposed to total submergence. Cytokinin accumulation was accompanied by phenotypic adaptations, including chlorophyll retention and increased biomass and carbohydrate content relative to WT plants. Abscisic acid accumulated rapidly in WT and IPT plants under waterlogging stress but remained low in all genotypes exposed to total submergence. IPT plants showed improved recovery after waterlogging stress was removed. Expression of ipt in submerged plants did not result in cytokinin accumulation until submergence stress was removed. At that point, IPT plants accumulated greater quantities of cytokinin and recovered to a greater extent than WT plants. This study established the relationship between flooding tolerance and cytokinin accumulation in IPT plants and suggested that translation of ipt transcripts and subsequent cytokinin accumulation were delayed under submergence stress.
Key words: Abiotic stress, abscisic acid, cytokinin, ipt gene, real-time RT-PCR, SAG12 gene, submergence, waterlogging
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Introduction |
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Flooding is a severe constraint on crop growth and affects about 16% of production areas worldwide (Boyer, 1982
). Soil can become flooded due to poor drainage or excessive rainfall or irrigation. The severity of the flooding stress is affected by flooding duration, crop variety, growth stage, soil type, fertility levels, pathogens, and flooding conditions (Sullivan et al., 2001). Flooding stress is initiated by either waterlogging, when only the roots are flooded, or complete submergence when the entire plants are under water. While some plants have adaptive mechanisms that permit survival during long-term waterlogging (Bacanamwo and Purcell, 1999a), most plants die within 1 or 2 d of submergence (Sullivan et al., 2001).
Flooding induces or accelerates plant senescence in tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum L.), sunflower (Helianthus annuus L.), barley (Hordeum vulgare L.), peas (Pisum sativum L.), wheat (Triticum aestivum L.), maize (Zea mays L.), and soybean (Glycine max L.) (Burrows and Carr, 1969; Drew and Sisworo, 1977; Olymbios and Schwabe, 1977; Jackson, 1979; Trought and Drew, 1980; VanToai et al., 1994). The most obvious symptom of flooding injury is leaf chlorosis, which is followed by necrosis, defoliation, cessation of growth, and premature plant death. Within 1 d of flooding, the concentration of the anti-senescence hormone, cytokinin, in sunflower xylem sap declined sharply to a very low level which may have resulted from the decreased cytokinin synthesis in roots and the inability of the anaerobic roots to export cytokinin to the aerial part of the plant (Burrows and Carr, 1969). Cytokinin is synthesized at the root apical meristem (Short and Torrey, 1972) where depressed metabolic activity and cell death due to flooding occur much earlier than in other tissues (VanToai et al., 1995).
To determine whether enhanced endogenous cytokinin production improved flooding tolerance, Zhang et al. (2000) generated transgenic plants containing the chimeric SAG12:ipt gene. The ipt gene encodes the enzyme isopentenyl transferase which catalyses the initial, rate-limiting step in the cytokinin biosynthesis pathway. The production of cytokinin in the SAG12:ipt transgenic plants is autoregulated because the senescence-specific SAG12 promoter was used to drive the expression of the ipt gene (Gan and Amasino, 1995). The prevention of senescence, in turn, attenuates promoter activity and prevents hormone overproduction. The SAG12:ipt (IPT) Arabidopsis plants showed normal growth but exhibited a 7–10 d delay in plant senescence. They remained greener and produced more biomass and seeds than wild-type (WT) plants under waterlogging and submergence stress (Zhang et al., 2000). Since cytokinin concentration was not determined in this study, the relationship between cytokinin concentration and flooding tolerance of IPT plants remains to be confirmed.
In many cases, plants that survive flooding die after the stress is removed (Sullivan et al., 2001). The post-flooding period can be as injurious as flooding itself, in part because of senescence-associated processes initiated in response to the original stress. Plants which are tolerant to flooding need to survive or grow during the stress but also to recover after the stress is removed. Most literature on flooding tolerance has focused exclusively on the flooding period and has neglected the post-flooding period.
In the current study, the comprehensive role of cytokinin in regulating flooding tolerance was characterized in IPT Arabidopsis plants at different time points during the 5 d of submergence and waterlogging stress and the 5 d post-flooding recovery period. The relationship of flooding tolerance responses to expression of the ipt and SAG12 genes was also documented, together with the accumulation of abscisic acid (ABA) during these periods.
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Materials and methods |
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Plant materials
Transformation of Arabidopsis thaliana ecotype Columbia plants with the Agrobacterium tumefaciens strain (GV 3101) containing the SAG12:ipt chimeric gene in a binary vector was performed as described by Zhang et al. (2000)
. Two independent transgenic lines (IPT3 and IPT5) were used in this study. Transgenic seedlings resistant to kanamycin were transplanted to 6 cm plastic pots containing Metro-Mix 350 (WR Grace & Co., Cambridge, MA, USA) medium at one plant per pot. Plants were grown in a growth chamber maintained at constant 24 °C and 16 h light (250 µmol photon m–2 s–1) for 2 weeks before being subjected to flooding stress. At that time, plants had 11 or 12 rosette leaves.
Flooding and recovery treatments
Transgenic (IPT3 and IPT5) and WT plants were subjected to complete submergence or waterlogging treatment for 1, 3, and 5 d. The submergence treatment consisted of submerging the entire plant under 6 cm of water, while in the waterlogging treatment the water was maintained at 1 cm above the soil surface. In the recovery experiment, the water was drained after 1, 3, and 5 d of submergence or waterlogging stress and the plants allowed to recover for 5 d. Control plants were not flooded. At each time point, total biomass and chlorophyll contents of seven individual plants were quantified. Soluble sugar, cytokinin, and ABA content and gene expression were analysed on pooled samples of seven plants.
Phenotypic analysis of responses to flooding and post-flooding recovery
Analysis of shoot biomass:
Above-ground parts collected from individual plants at each time point were immediately frozen with liquid nitrogen and freeze-dried. Dry tissues were ground, weighed, and stored in tightly sealed plastic vials.
Analysis of chlorophyll:
Leaf discs (3 mm diameter) were harvested from each of the three fully expanded uppermost leaves, soaked in 3 ml dimethylformamide (DMF) and incubated in the dark for 48 h at 4 °C. Absorbance of the DMF extract was measured at 647, 665, and 720 nm with a dual-beam spectrophotometer (Beckman DU-50 Series, Fullerton, CA, USA) using 100% DMF as a blank. Chlorophyll concentration was calculated in mg l–1 using the equation of Inskeep and Bloom (1985) modified from the formulation of Moran (1982).
Analysis of soluble sugars:
Sucrose, glucose, and fructose of harvested plant tissues were determined as described by Streeter and Strimbu (1998). Soluble sugar was extracted from dried, ground tissues (5 mg) with 125 µl pyridine and 125 µl STOX reagent (Pierce Chemical Co., Rockford, IL, USA). The mixtures were vortexed and incubated at 70 °C for 40 min with occasional mixing. The mixtures were then cooled to room temperature and thoroughly mixed with 200 µl hexamethyldisilazane and 20 µl trifluoroacetic acid. After an additional 60 min incubation at room temperature, soluble sugar concentrations were determined using a Hewlett-Packard 5890 Series II gas chromatograph (Hewlett-Packard Co., Avondale, PA, USA) equipped with a 3% OV-17 on Chromsorb WHP column. Peak areas were quantified with a Hewlett-Packard 3396A integrator. All samples were analysed twice.
Analysis of cytokinin and ABA:
Lyophilized leaf samples were extracted with chilled 80% methanol, cytokinins present in the extracts were purified by reverse-phase high-performance liquid chromatography, and zeatin, dihydrozeatin, zeatin riboside (ZR), dihydrozeatin riboside, isopentenyl adenine, isopentenyl adenosine (iPA), and ABA content were quantified as previously described (Trione et al., 1985; Banowetz et al., 1994). Total cytokinin was calculated as the sum of individual components.
Analysis of gene expression by the real-time RT-PCR:
Total RNA was extracted from lyophilized leaf samples by the lithium chloride method (Franz and Wolfgang, 1993). First-strand cDNA synthesis was performed using the Ready-To-Go T-Primed First-Strand Kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA). Real-Time PCR was performed with specific primers of the ipt gene (forward 5'-ACCCATGGACCTGCATCTA; reverse 5'-GGAGCTCAGGGCGGCGTAACC) and of the native SAG12 genes (forward 5'-GTGTCTACGCGGATGTGAAG; reverse 5'-CAGCAAACTGATTTACCGCA) by the intercalating SYBR Green method using the ABI Prism 7900HT Sequence Detection System (Applied Biosystem, Foster City, CA, USA). Gene expression was quantified with the standard curve of actin gene (forward 5'-CTGGAGATGATGCACCAAGA; reverse 5'-CCTCATCACCAACGTAAGCA) amplified from a serial dilution of control, non-flooded samples using the manufacturer's protocol (Ambion, Inc., Austin, TX, USA). Relative expression values were normalized to the lowest expression value taken as 1. Samples were run in duplicate, and data presented are the mean of the two replications ±standard deviation. The presence of a single amplicon of the predicted size was confirmed by melting curve analyses of the data.
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Results |
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Biomass analysis
Transgenic IPT plants generally accumulate 20–30% more shoot biomass than WT plants under non-stress control conditions (Zhang et al., 2000
). To avoid the potential effects of individual differences in plant vigour on flooding tolerance, the plants in the current study were selected to have the same size. Consequently, no difference in biomass accumulation was detected between WT and IPT3 and IPT5 transgenic plants at each of three sampling times in the non-flooded control treatment (Table 1). One day of submergence showed no damage on growth of both WT and IPT plants. Both genotypes, however, lost about 10% biomass at day 3. IPT plants quickly adapted to the stress and resumed growth. At day 5, IPT plants contained 15% more biomass than plants at day 3, while WT plants showed no growth during submergence.
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It is interesting that waterlogging was not damaging to the growth of Arabidopsis plants. Both WT and IPT plants of the 1 d waterlogging treatment accumulated more biomass than control plants of the same age. After 3 d of waterlogging treatment, transgenic plants accumulated more biomass than control plants, while WT plants grew as well as control plants. The IPT plants subjected to the 5 d waterlogging treatment continued to grow as well as control plants, while WT plants accumulated 20% less biomass.
In the recovery experiment, plants were removed from the control, submergence, and waterlogging treatments at day 1, day 3, and day 5, and allowed to recover for 5 d before biomass accumulation was quantified. As expected, control, non-flooded WT, IPT3, and IPT5 plants accumulated 60–140% more biomass during the additional 5 d of growth but no difference was detected between the genotypes (Table 2). The results confirmed that IPT plants grow normally under control, non-stressed conditions compared with WT plants. In the submergence treatment, no difference was detected between WT and IPT plants recovering from 1 d stress. IPT plants subjected to 3 d and 5 d submergence treatment recovered and grew significantly better than WT plants that received the same stress treatment (Table 2). Similarly, no difference in biomass was detected between WT and IPT plants recovered from 1 d of waterlogging. As waterlogging became more severe, the differences between IPT and WT plants became more distinct. In the 3 d and 5 d treatments, recovering IPT plants accumulated 11% and 25% more biomass than WT plants receiving the same stress duration, respectively (Table 2).
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The results showed that IPT plants grew better than WT plants under submergence and waterlogging stress. When the stress was removed, transgenic plants recovered faster and accumulated more biomass than WT plants.
Chlorophyll analysis
No difference in the chlorophyll concentration was detected between non-flooded WT and IPT plants. At day 1 of the non-flooded treatment, the average concentration was 689±48 µg g–1 FW. As the plants matured, the concentration increased to 738±9 µg g–1 FW at day 3 and 825±23 µg g–1 FW at day 5. Waterlogging for 1 d did not change the chlorophyll concentration of WT and IPT plants (718±25 µg g–1 FW) as compared with non-flooded control plants. After 3 and 5 d of waterlogging stress, the average chlorophyll concentration was 10% higher in IPT (745±24 µg g–1 FW) than in WT plants (678±38 µg g–1 FW). Submergence stress reduced chlorophyll concentration of both WT and transgenic plants by 19% at day 3 (593±8 µg g–1 FW) and 35% (538±10 µg g–1 FW) at day 5.
In the post-flooding recovery phase, IPT plants contained 5–8% more chlorophyll content during recovery from waterlogging stress and 20% more chlorophyll during recovery from 5 d of submergence than WT plants that received the same treatment.
Soluble sugar analysis
Control, non-flooded WT and IPT plants contained similar levels of fructose (3–9 µg mg–1 DW). Fructose levels decreased to c. 2 µg mg–1 DW in all genotypes at day 1 of the submergence treatment and remained essentially unchanged at day 3 and day 5 (Fig. 1A). Fructose levels did not change at day 1 of waterlogging stress in all genotypes. At day 3 and day 5, waterlogged plants accumulated five to seven times more fructose compared with control plants; but no consistent pattern was detected between the genotypes (Fig. 1A).
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The glucose levels were also similar in control WT and IPT plants (16–21 µg glucose mg–1 DW). At day 1 of submergence stress, the glucose levels were depressed to 50% of the controls and remained essentially unchanged at day 3 and day 5 (Fig. 1B). Contrary to submergence stress, WT and IPT plants accumulated 35–150% more glucose at day 1 of the waterlogging stress compared with the control. These levels continued to increase at day 3 and day 5 to 5-fold and 10-fold more than the controls, respectively (Fig. 1B).
WT and IPT plants contained an average of 24 µg sucrose mg–1 DW at day 1 of the control treatment. At day 3, sucrose levels increased to 50 µg mg–1 DW. The level remained unchanged at day 5 (Fig. 1C). At day 1 of submergence stress, the sucrose levels were depressed to 10 µg mg–1 DW and remained essentially unchanged at day 3 and day 5 of submergence. Sucrose levels remained unchanged at day 1 of waterlogging stress and began to increase up to 3-fold at day 3. No difference in sucrose levels was detected between the genotypes until day 5 of waterlogging when IPT plants contained 60% more sucrose (230 µg mg–1 DW) than WT plants (140 µg mg–1 DW).
In the recovery study, no consistent pattern in fructose, glucose, and sucrose accumulation was detected that differentiated IPT from WT plants (Fig. 2). Generally, the levels of all three sugars increased 4–5-fold in the recovery from submergence stress; sugar levels did not change much in the recovery from 1 d of waterlogging stress, but declined in the recovery from 3 and 5 d of waterlogging stress.
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Cytokinin analysis
The average cytokinin concentration in shoot tissue of control, non-flooded WT plants was 100 ng g–1 DW, while levels measured in IPT3 and IPT5 plants ranged from 250 to 300 ng g–1 DW (Fig. 3A). At day 1 of submergence, cytokinins decreased significantly in all genotypes, although the levels were still 2-fold higher in IPT3 and IPT5 plants (100 ng g–1 DW) compared with WT plants (40 ng g–1 DW) (Fig. 3A). At day 3 and day 5 of submergence, cytokinin levels were similar in WT and transgenic plants. At day 1 of waterlogging, cytokinin levels declined from 50% to 100% in all genotypes (68–108 ng g–1 DW) compared with non-flooded control plants (Fig. 3A). At day 3 of waterlogging, cytokinin levels further declined in WT plants (26 ng g–1 DW), but increased 2–4-fold in IPT5 and IPT3 plants (350–1500 ng g–1 DW). At day 5 of waterlogging, cytokinin levels increased substantially in WT plants (700 ng g–1 DW), but remained 50% lower than levels measured in IPT3 and IPT5 plants (1400–1500 ng g–1 DW) (Fig. 3A).
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In the recovery experiment, cytokinin levels in IPT plants were consistently higher than in WT plants. IPT plants subjected to 3 d submergence stress accumulated up to 10-fold more cytokinin (700 ng g–1 DW) than WT plants (70 ng g–1 DW). A 2-fold higher cytokinin accumulation was also detected in IPT plants recovered from 5 d submergence stress (1800 ng g–1 DW) compared with WT plants stressed for the same duration (850 ng g–1 DW) (Fig. 3A).
Similarly, IPT plants that recovered from waterlogging stress consistently accumulated more cytokinin than WT plants that received the same stress treatment. IPT plants recovered from the 1 d waterlogging stress contained 150–180 ng g–1 DW while WT plants only contained 80 ng g–1 DW. After recovery from the 3 d waterlogging treatment, cytokinin levels remained at 50 ng g–1 DW in WT, but increased to 375 and 700 ng g–1 DW in IPT3 and IPT5 plants, respectively. After recovery from the 5 d waterlogging treatment, cytokinin levels were much lower in WT plants (200 ng g–1 DW) compared with IPT3 (350 ng g–1 DW) and IPT5 plants (750 ng g–1 DW).
Differences in cytokinin levels between WT and IPT plants were due largely to changes in ZR content (Table 3). Under control, non-flooded conditions, ZR-type cytokinins were the predominant form in WT plants (c. 95%), while the iPA-type cytokinin only contributed 4% of the total (Table 3). This iPA:ZR ratio of 0.04 was similar in control IPT plants despite the fact that transgenics accumulated twice as much cytokinin. As WT plants in this study aged, the iPA:ZR ratio increased to 0.28 at day 5 of the control treatment (Table 3). Under submergence stress, the iPA:ZR ratio was as large as 1.20 suggesting that the enzyme(s) converting iPA to ZR were affected by senescence and severe stress conditions. The much lower iPA:ZR ratio of IPT plants under submergence and waterlogging treatments could be interpreted as another indication of their tolerance to the stresses.
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Abscisic acid analysis
Average ABA concentration in shoot tissue of non-flooded plants of all genotypes ranged from 10 to 20 ng g–1 DW (Fig. 4A). Under submergence stress, ABA levels were reduced substantially in all genotypes. By contrast, ABA levels increased 1.5–3-fold in all genotypes under waterlogging stress and continued to increase at 3 d and 5 d to as high as 700 ng g–1 DW (Fig. 4A). There was no consistent pattern that suggested IPT plants accumulated more ABA than WT plants.
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In the recovery experiment, WT and IPT differed in ABA accumulation only in plants exposed to 5 d of submergence (Fig. 4B). Similarly, significant genotypic differences during recovery from waterlogging were only apparent in plants exposed to 5 d of waterlogging.
Gene expression
Relative expression of the ipt transgene, the native SAG12 gene and the positive control actin gene was quantified by real-time RT-PCR. Expression of the actin gene remained essentially constant in all treatments. Ipt transcripts were detected in control, non-flooded IPT3 and IPT5 plants at levels 50-fold and 250-fold higher than in control WT plants, respectively (Fig. 5A). After 1 d of submergence, ipt gene expression increased 100-fold in IPT3 plants and 25-fold in IPT5 plants. Ipt transcript levels increased at day 3 and day 5 of submergence to c. 250-fold in IPT3 plants and c. 500-fold in IPT5 plants. The increase in ipt transcript accumulation in IPT plants under submergence was highly significant, but even greater increases occurred in plants subjected to waterlogging stress. At 1 d of waterlogging, ipt-gene expression increased c. 400-fold in IPT3 and IPT5 plants. The accumulation was further increased at day 3 and day 5 to 1500- and 2000-fold, respectively, in IPT3 plants. In IPT5 plants, the increase was as high as 4000-fold at day 5 of waterlogging (Fig. 5A).
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Expression of the senescence-specific, native SAG12 gene was at base-line levels in non-flooded plants of both genotypes. In response to flooding stress, SAG12 gene expression followed an almost opposite pattern to that of the ipt gene. The expression remained at base line in IPT plants throughout the entire submergence duration, but increased 5-fold in WT plants at the day 5 treatment. Under waterlogging, SAG12 gene expression of IPT3 and IPT5 plants remained at base-line levels at day 1 and day 3 of waterlogging stress. At day 5, the expression increased 10-fold in IPT plants. WT plants showed a 5-fold increase in SAG12 expression at day 1 of waterlogging. The expression increased to 10-fold at day 3 and 1800-fold at day 5.
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Discussion |
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Phenotypic responses of IPT plants to waterlogging and submergence stresses
The fact that IPT plants are more tolerant of submergence and waterlogging than WT plants can further be substantiated by the results of similar studies conducted under different conditions at two other locations. In all these studies, the similar trend that transgenic plants grew bigger than WT plants at 5 d of submergence and 3 d and 5 d of waterlogging was observed (data not shown). In addition, the differences between transgenic and WT plants would be much greater if random IPT plants were assigned to the treatments. As mentioned, to avoid the confounded effects due to the generally 20–30% larger size of IPT plants compared with WT plants, IPT plants of similar size to WT plants were selected for the treatments. Consequently, the selected IPT plants were smaller than average IPT plants.
Arabidopsis plants are more tolerant to waterlogging than other dicots (Burrows and Carr, 1969; Jackson, 1979). According to Scott et al. (1989), waterlogging for as little as 2 d reduced soybean grain yield from 18% to 26% depending on the plant growth stage. In the present study, 5 d of waterlogging reduced biomass of WT Arabidopsis by only 12% and did not affect biomass of IPT plants (Table 1). The results also showed that submergence was more injurious to Arabidopsis than root waterlogging. Plants subjected to waterlogging develop mechanisms to transport oxygen from the shoot to the root, but plants subject to submergence were exposed completely to a low or anoxic environment and low light intensity (Jackson and Ricard, 2003). Arabidopsis plants also were more tolerant of submergence than soybean plants which were completely killed by 3 d of submergence (Sullivan et al., 2001). Studies using biomass, machine vision, and image processing to quantify growth response of soybeans to waterlogging stress showed a two-phase response: the first ‘reactive’ phase which occurred immediately after the stress, when plants grew very little or not at all, was followed by a second ‘acclimatized’ phase, when growth resumed (Bacanamwo and Purcell, 1999b; VanToai et al., 2003). The duration of the reactive phase was shorter in the flooding-tolerant genotypes than the flooding-susceptible genotypes, indicating that flooding-tolerant genotypes acclimatized more quickly to stress than flood-susceptible genotypes. It is interesting that an apparent two-phase response in biomass accumulation occurred during submergence stress in this study where the reactive phase was 2 d shorter in IPT than in WT plants (Table 1). The two-phase response was not obvious in the waterlogged plants where both WT and IPT Arabidopsis plants appeared tolerant.
Tolerance of IPT plants to submergence and waterlogging stresses was also observed during the post-flooding recovery phase when the transgenic plants accumulated more biomass than WT plants. The capacity of IPT plants to accumulate greater amounts of biomass under waterlogging and submergence stresses, as well as during recovery from stress, suggests a potential approach to improve flooding tolerance of crop plants.
The shortage of oxygen associated with flooding stress has been implicated as the primary cause of flooding injuries. Indeed, anoxic and hypoxic stress have been used synonymously with flooding stress. The metabolic basis of injury from anoxia is generally attributed to the imbalance between ATP supply and demand (Jackson and Ricard, 2003). The inefficient ATP production by the anaerobic fermentation pathway requires a continuous supply of glucose to fuel glycolysis. Lack of glucose and sucrose to support glycolysis has been recognized as the main cause of injury and death of flooded plants (Saglio, 1985; Perata et al., 1997). Recently, Huang et al. (2003) reported that at day 3 of anoxia, the sugar concentrations in embryos and endosperms of the tolerant rice (Oryza sativa L.) genotype Amaroo was nearly 4-fold higher than in the intolerant IR22 genotypes. In the present study, IPT plants accumulated more sucrose than WT plants under waterlogging stress and during recovery from flooding. Whether the increase in sucrose accumulation of transgenic plants is the causal mechanism or the consequence of flooding tolerance remains unclear. The severity of submergence stress compared with waterlogging was also demonstrated by the decline of soluble sugar in submerged plants, while sugars continued to increase in plants under waterlogging stress. However, unlike transgenic SAG12:ipt tobacco plants, which accumulated 3–5-fold more glucose and fructose during senescence than WT plants (Wingler et al., 1998), no genotypic difference in the accumulation of these sugars was detected under submergence, waterlogging, and post-flooding recovery periods.
An indicator of flooding-associated injury accompanying premature senescence is leaf chlorophyll content (Daugherty and Musgrave, 1994). The tolerance of Arabidopsis plants to waterlogging stress suggested by biomass accumulation in this study was also supported by the leaf chlorophyll content. WT plants subjected to submergence and waterlogging had more yellow and purple coloration than IPT plants (Fig. 6). Leaf samples for chlorophyll measurement in this study were taken from the uppermost leaves. Since significant differences in senescence progress between WT and IPT plants were only observed in older leaves (Gan and Amasino, 1996, 1997; McCabe et al., 2001), the higher chlorophyll concentration detected in transgenic plants subjected to the waterlogging treatment and the post-waterlogging and post-submergence recovery phase could have been even more distinct if samples were collected from the entire plant.
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The elevated chlorophyll levels in IPT plants subjected to waterlogging could have resulted from inhibition of chlorophyll breakdown as well as the accumulation of its newly synthesized products. Indeed, cytokinin treatment increases
-aminolevulinic acid synthase activity involved in chlorophyll synthesis (Fletcher et al., 1973; Fukuda and Toyama, 1982; Purohit, 1982).
Accumulation of cytokinin in IPT plants in response to waterlogging and submergence
Elevated chlorophyll content of IPT plants under waterlogging stress was correlated with elevated leaf cytokinin content. The premature senescence in plants exposed to flooding stress has been attributed to decreased cytokinin synthesis in roots and its transport to shoots (Neuman et al., 1990). Measurement of the temporal pattern of cytokinin accumulation in this study revealed a correlation between elevated cytokinin levels and flooding tolerance of IPT plants. The initial decline in cytokinin levels after 1 d of waterlogging and submergence in both WT and IPT plants was similar to the results reported in xylem sap of flooded sunflower (Burrows and Carr, 1969). As waterlogging continued, cytokinin levels increased in both genotypes, but the increase occurred 2 d earlier and attained a greater magnitude in transgenic plants than in WT plants, reflecting the induction of ipt gene expression by the senescence-specific SAG12 promoter. The apparent initial decrease in cytokinin content followed by increased accumulation in response to waterlogging suggests a two-phase response. It is intriguing that the accumulation of biomass under waterlogging did not show that two-phase response.
Accumulation of ipt and SAG12 transcripts in IPT plants in response to waterlogging and submergence
While roots are the major site of cytokinin synthesis, expression of the GUS reporter gene driven by the SAG12 promoter is not detected in roots (Zhang et al., 2000). The high levels of cytokinin in IPT plants associated with the increased ipt transcript accumulation indicated that leaf expression of the ipt gene produced a functional isopentenyl transferase enzyme. Of special note was the observation that ipt transcript accumulation under submergence stress did not result in increased cytokinin accumulation. The lack of relationship between ipt transcript accumulation and cytokinin accumulation during submergence stress suggests that either translation of the ipt transcript was impacted, or the activity of the translated product was inhibited, possibly due to limiting quantities of oxygen and adenylate energy. Alternately, cytokinins could be less stable or metabolized more rapidly under submergence stress.
The SAG12 gene was first identified from leaves that were in the late stage of senescence and were visibly yellow (Weaver et al., 1998). In the present study, the large increase in SAG12 transcript accumulation in WT plants at day 5 of submergence and waterlogging treatments indicated the association between senescence and stress in WT plants, whereas no accumulation was detected in IPT plants. As expected, the temporal pattern of SAG12 gene expression was opposite to that of the ipt gene, showing the effectiveness of cytokinin in delaying senescence.
Accumulation of ABA in IPT plants in response to waterlogging and submergence
Flooding can induce changes in hormones other than cytokinins, including ABA. According to Hwang and VanToai (1991), exogenous treatment with ABA improved anoxic tolerance of corn seedlings. Chang et al. (2003) reported that ABA regulated floral senescence and its accumulation was significantly higher in corollas of WT than of IPT petunia. In the present study, ABA and cytokinin accumulation increased simultaneously. The increased ABA accumulation during waterlogging was similar to the results reported in pea plants (Zhang and Davies, 1990). Since flooded plants suffer from water deficit due to the decreased hydraulic conductance caused by the shortage of oxygen and energy charge (Else et al., 2001), the increase in ABA can be taken as an adaptive measure that regulates stomatal closure to avoid the leaf wilting which commonly occurred in flooded plants. Additionally, ABA may increase the growth stimulatory effect of cytokinin as reported by Blumenfield and Gazit (1970).
This study identified and characterized impacts of cytokinin overproduction on the tolerance of Arabidopsis plants to waterlogging and submergence stresses. The increased tolerance of both transgenic IPT3 and IPT5 lines to waterlogging coincided with the increase in cytokinin levels during the flooding and recovery periods. IPT plants accumulated more biomass and more cytokinin after 1 d submergence stress. However, the increases in ipt transcript accumulation at day 5 of this treatment were not accompanied by cytokinin accumulation. IPT plants also accumulated more cytokinin and showed improved recovery from the submergence stress than WT plants. These results suggest that cytokinin accumulation during waterlogging and recovery from waterlogging and submergence is an important mechanism of flooding tolerance.
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Acknowledgements |
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We are grateful to Mr Bert Bishop of the Ohio Agricultural Research and Development Center, Ohio State University, Wooster, OH, USA for his help with statistical analysis of the data. This paper (manuscript number HCS 03-29) is a joint contribution of the USDA-ARS and Ohio State University. Partial salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.
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Footnotes |
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Use of trade names is for the benefit of readers and does not imply endorsement of the product by the US Department of Agriculture.
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References |
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