Modulation of spermidine and spermine levels in maize seedlings exposed to long-term salt stress. In this study, we measured polyamine content in maize leaves of semi-hydroponically grown seedlings exposed to 1 and 7 days of salt stress. The effect of salt stress on the expression of the main genes involved in polyamine biosynthesis was also assessed.
Our data show a time- and NaCl-dependent regulation of the Zmspds2 and Zmspds1 genes, suggesting that the former may be hyperosmotically responsive while the later is NaCl responsive. Transcriptional regulation of some polyamine biosynthetic genes, mainly adc , has been reported under salt stress. The physiological status of maize seedlings subjected to salt stress (see Materials and Methods) was assessed considering two parameters: the levels of proline (Pro), as a universal marker of water deficit and salinity [22, 42], and the chlorophyll -content in stressed leaves.
Effect of salt stress on the expression of the Zmspds1, Zmspds2, adc and samdc genes. The maize Zmspds2 gene can be transcribed into two known splice variants, Zmspds2A and Zmspds2B, under salt stress conditions [37]. We also measured the expression of the adc gene, responsible for Put biosynthesis, and observed that the transcript levels were down-regulated at 25 and 150 mM at both 1 and 7 days of stress (Figure 3D,I).
In this sense, our data show a time- and NaCl-dependent regulation of the Zmspds2 and Zmspds1 genes, suggesting that the former could be hyperosmotic responsive, while the later could be NaCl responsive.
Discussion
The differential regulation of both maize spds genes could play an important role in the proper adjustment of polyamine levels under stress. In addition to the spermidine synthase genes, the maize samdc gene was also up-regulated after 1 day of salt stress. Changes in the samdc gene expression pattern have been reported during different stages of plant development [30] and in response to abiotic stress conditions.
In the case of the adc gene, no obvious changes in the expression pattern were observed, although many reports support the participation of this gene in the response to plant salt stress. Usually, increases in gene expression during stress treatment do not show a direct relationship with polyamine accumulation, as observed in our study in the case of adc transcription and Put levels. We suggest the existence of some post-translational regulation mechanisms of ADC as described in other plant systems [ 36 , 38 ], although we do not rule out the existence of other adc genes in the maize genome as described for rice ( GRAMENE: A Map Comparative Cereal Resource, USA), and that analyzed here is not induced in response to salt stress.
In the case of maize spermidine synthases, the active participation of these genes takes place while the plant tends to maintain or accumulate Spm. As described in Results, marked increases in Spd and Spm levels were observed at 25 mM NaCl on day 7, while slight increases were observed at all NaCl concentrations applied at 1 day of stress, linking the transcriptional data with those from accumulation. 27] reported that salt stress elicits endogenous ABA accumulation, which may act as a signal for PA accumulation under stress.
Our data suggest that ABA may act as an important signaling molecule in the induction of PA biosynthesis at the transcriptional level with the subsequent maintenance of the PA pool (especially Spm) in maize leaves under salt stress. Many authors support the idea that tolerant cultivars mainly accumulate Spd and Spm compared to the sensitive ones that accumulate. Place under salt stress [25, 40]. We compared the pattern of PA accumulation with that of proline, and observed some differences in the time point at which biosynthesis was induced and also the concentration reached under salt stress.
Although the accumulation of PAs was not as dramatic as that of Pro in maize, salt stress rather stimulated the biosynthesis of PAs. In addition to Pro, we measured the total chlorophyll content in the salt-stressed maize seedlings. Accordingly, we observed a reduction in total chlorophyll content when stress became severe on day 14.
Methods 1. Plant Material
Generally, 300 mg of the plant material was extracted with 1 mL of 5% (v/v) perchloric acid (PCA) and then incubated overnight at 4 C. The extracts were recovered by centrifugation and 200 µL of the supernatants were dansylated in a mixture containing 100 µL saturated Na2CO3, 200 µL dansyl chloride (5 mg.mL-1 acetone) and 5 µL 100 D-diamino-heptane (HT 1-7) contains ) as internal standard. One L of the RT reaction was used as template for PCR assays, which amplify the adc, samdc, Zmspds2 and actin-1 gene transcripts [37].
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33] Perez-Amador M.A., Leon J., Green P.J., Carbonell J., Induction of the arginine decarboxylase gene ADC2 provides evidence for polyamine involvement in the Arabidopsis wound response. Ten L of RT-PCR product was loaded onto each lane and separated by electrophoresis on a 1% (w/v) agarose gel. In case A' and F', 20 L of PCR product was loaded to visualize the Zmspds2B splice variant.
Ten L of the RT-PCR product was loaded onto each lane and separated by electrophoresis on 1% (w/v) agarose gel.
