Dataset Information

High cytokinin levels induce a hypersensitive-like response in tobacco.

ABSTRACT:

Background and aims

Cytokinins are positive regulators of shoot development. However, it has previously been demonstrated that efficient activation of the cytokinin biosynthesis gene ipt can cause necrotic lesions and wilting in tobacco leaves. Some plant pathogens reportedly use their ability to produce cytokinins in disease development. In response to pathogen attacks, plants can trigger a hypersensitive response that rapidly kills cells near the infection site, depriving the pathogen of nutrients and preventing its spread. In this study, a diverse set of processes that link ipt activation to necrotic lesion formation were investigated in order to evaluate the potential of cytokinins as signals and/or mediators in plant defence against pathogens.

Methods

The binary pOp-ipt/LhGR system for dexamethasone-inducible ipt expression was used to increase endogenous cytokinin levels in transgenic tobacco. Changes in the levels of cytokinins and the stress hormones salicylic, jasmonic and abscisic acid following ipt activation were determined by ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS). Trends in hydrogen peroxide content and lipid peroxidation were monitored using the potassium iodide and malondialdehyde assays. The subcellular distribution of hydrogen peroxide was investigated using 3,3'-diaminobenzidine staining. The dynamics of transcripts related to photosynthesis and pathogen response were analysed by reverse transcription followed by quantitative PCR. The effects of cytokinins on photosynthesis were deciphered by analysing changes in chlorophyll fluorescence and leaf gas exchange.

Key results

Plants can produce sufficiently high levels of cytokinins to trigger fast cell death without any intervening chlorosis - a hallmark of the hypersensitive response. The results suggest that chloroplastic hydrogen peroxide orchestrates the molecular responses underpinning the hypersensitive-like response, including the inhibition of photosynthesis, elevated levels of stress hormones, oxidative membrane damage and stomatal closure.

Conclusions

Necrotic lesion formation triggered by ipt activation closely resembles the hypersensitive response. Cytokinins may thus act as signals and/or mediators in plant defence against pathogen attack.

SUBMITTER: Novak J

PROVIDER: S-EPMC3690983 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:HpaXm produced by the cotton leaf blight bacterium Xanthomonas citri subsp. malvacearum is a novel harpin elicitor of the induced hypersensitive response (HR) in tobacco. We investigated whether fragments of HpaXm, compared with fragments of Hpa1Xoo, are sufficient for HR or plant growth promotion (PGP) elicitation using four synthetic peptides (HpaXm35-51, HpaXm10-39, Hpa1Xoo36-52 and Hpa1Xoo10-40). We also heated the fragments to determine the heat tolerance of the functional fragments. HpaXm35-51 and Hpa1Xoo36-52 induced hypersensitive response (HR). Bursts of reactive oxygen intermediates (ROI) induced by HpaXm35-51 and Hpa1Xoo36-52 were earlier and stronger than those induced by HpaXm and Hpa1Xoo. In plants treated with HpaXm35-51 or Hpa1Xoo36-52, the expression of the HR marker genes Hin1 and Hsr203J and the active oxygen metabolism related gene AOX were significantly upregulated. These findings suggest that the predicted α-helical structures of the HpaXm35-51 and Hpa1Xoo36-52 fragments are crucial for HR. PGP result by soaking seeds in unheated/heated HpaXm10-39 or Hpa1Xoo10-40 solution prior to transfer, which obviously enhances root growth and the aerial parts of plants. The PGP related gene NtEXP6 was greatly enhanced when plants were sprayed with a solution of HpaXm10-39 or Hpa1Xoo10-40; heated fragment treatments induced higher levels of NtEXP6 expression than unheated HpaXm fragments. In addition, HR marker genes induced by the heated fragments had lower expression levels than when induced with unheated HpaXm fragments. Moreover, the expression levels of HR marker genes and PGP related genes induced by treatment with Hpa1Xoo fragments before or after heating were the opposite of those induced by HpaXm fragments. Different functional fragments of harpin and different harpins with the same functional region have different degrees of heat tolerance. Therefore, the heat resistance of harpin is conservative, but the degree of heat tolerance of the functional fragments is specific.