MEDICI, L. O.

SANTOS, M. P.

ZANDONADI, D. B.

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,QWHUQDWLRQDO6FLHQWL¿F&HQWUHRI)HUWLOL]HUV&,(&Rio de Janeiro, RJ 2014PROCEEDINGS9LQLFLXVGH0HOR%HQLWHV$GLOVRQGH2OLYHLUD-XQLRU3DXOR6HUJLR3DYLQDWR3DXOR&pVDU7HL[HLUD0LOWRQ)HUUHLUD0RUDHV5HJLQD0DULD9LOODV%{DVGH&DPSRV/HLWH5RQDOGR3HUHLUDGH2OLYHLUDEditorsTECHNOLOGICAL INNOVATION FOR A SUSTAINABLE TROPICAL AGRICULTUREOctober 20-24, 2014Rio de Janeiro, RJ - Brazil16th World Fertilizer Congress of CIEC24434Introduction Increasing environmental constraints, such as salinity are threatening the productivity of crops worldwide. Near to 77 million hectares land is af-fected by this problem (FAO, 2007). The intensive use of soluble fertilizers is considered one of the causes of salinization. Either natural soil organic matter or humic substances has been reported as important to ameliorate both drought and salin-ity of soils due their indirect and direct effects (Bot DQG%HQLWHV0RUDHWDO 7KHHIIHFWVof salt stress may alter photosynthesis and cause oxidative stress. Humic Acids (HAs) isolated from vermicompost appear to restore water capacity of $%$GH¿FLHQWWRPDWRVLWLHQV, thus it is not surprising that HAs could affect plant physiology during abi-otic stress. In this work we evaluated vermicompost HAs as potential alleviator of salt stress in tomato plants. Pre-treatment with this synthetic organic material helped plants to cope with salt stress by a mechanism related to photosynthesis maintenance, membranes stability and the activity of nutrient up-take and salt stress related enzymes. It is postu-lated that HAs decreases the harmful effects of salt VWUHVV7RWKHEHVWRIRXUNQRZOHGJHWKLVLVWKH¿UVWwork showing that plasma membrane H+-ATPase is related to Na exclusion in NaCl stressed plants pre-treated with HAs and that humic substances can directly affect photosynthesis processes during stress. Methods Growth and salinization of plant material was performed as described by Binzel (1995) with VRPH PRGL¿FDWLRQV %ULHÀ\ DIWHU  GD\V XQGHUhalf strength Hoagland’s solution (HSHS), tomato (6RODQXP /\FRSHUVLFXP cv Rheinlands Ruhm, RR) plants were pre-treated with half strength Ho-agland’s solution only (con) or HSHS plus HAs for 4 days (HAs). Subsequently, plants were transferred to HSHS plus 200mM NaCl for additional 3 days FRQ!1D&O RU +$V!1D&O 3ODQWV ZHUH DQDO\]HGafterwards. Each treatment consisted of 10 plants. Experiments were repeated three times.Results and discussion Humic substances have been used to im-SURYH IHUWLOL]HUV HI¿FLHQF\ VLQFH LW FRXOG HQKDQFHPLQHUDO QXWULHQWV XSWDNH DQG UHGXFH QXWULHQW ¿[D-WLRQ LQVRLO (UURHWDO-DQQLQHWDOHumic substances isolated from different sources are able to increase proton pumps activity, appar-ently due its auxin-like effect and nitric oxide pro-duction (Zandonadi et al., 2010), but little is known about the role of these organic substances during plant stress. Tomato plants (cv. Rheinlands Ruhm), exposed to increasing salt concentration until 300 mM NaCl presented a decline of its initial indole 3-acetic acid (IAA) levels in the roots by nearly 75% (Dunlap and Binzel, 1996). The presence of IAA in the HAs structure was detected (Canellas et al., 2002) and here we show that the pre-treatment of tomato plants with humic acids (HAs) resulted in an alleviation of saline deleterious effects (Figs. 1 and 2). The mean photosynthetic rate, transpiration rate, the stomatal conductance and chlorophyll a content of tomato plants were reduced by exog-enous application of 200mM NaCl (Fig. 1). Mem-brane integrity and oxidative stress as measured by means of malondialdehyde (MDA) content were affected by NaCl and recovery due HAs pre-treat-ment was also observed (Fig. 1). Mitigation of salt stress by humic substances appears to be related to both direct and indirect features of photosynthe-sis. To date the only work showing effects of HAs on plant photosynthesis during salt stress concluded that there is no improvement in salt tolerance due HAs treatments (Liu and Cooper, 2002). It is hard to MITIGATION OF SALT STRESS OF TOMATO USING PRE-TREATMENT WITH HUMIC ACIDSDANIEL BASÍLIO ZANDONADI1, MIRELLA PUPO SANTOS2 LEONARDO OLIVEIRA MEDICI31 /DERUDWyULRGH%LRORJLD&HOXODU5RGRYLD%5.P%UDVtOLD$QiSROLV)D]HQGD7DPDQGXi&DL[D3RVWDO%UDVtOLD%5$=,/GDQLHO]DQGRQDGL#HPEUDSDEU 2 /DERUDWyULRGH7UDQVIRUPDomR*HQpWLFD3DUTXH(VWDomR%LROyJLFD3T(%$Y:1RUWH&DL[D3RVWDO%UDVtOLD%5$=,/PLUHOODSXSR#\DKRRFRPEU 3 Departamento de Ciências )LVLROyJLFDV%5NP6HURSpGLFD%5$=,/OPHGLFL#JPDLOFRP245compare the results from this study with the present work, since here we did a pre-treatement instead an addition of HAs during salt stress. In addition, plant metabolisms are very different in each case. The plant membrane transporters are cru-cial in increasing both nutrient content and resist-ance salinity stress. Binzel (1995) reported a stimu-lation of both plasma membrane and tonoplast AT-Pase proton pumps transcripts in tomato roots dur-ing 150-400 mM NaCl stress. In agreement, here HAs were able to enhance proton pumps activity prior to salt exposition, an important step for detoxi-fying Na+ ions from cytosol (Fig. 1). Auxin levels de-clines when plants are challenged with NaCl (Dun-lap and Binzel, 1995). The Has sample used here has IAA in its structure, thus IAA could be associ-ated to either a direct effect on plant proton pumps as endogenous hormonal balance favoring the salt tolerance observed. It is worthy to note that HAs are able to induce nitric oxide production (Zandonadi et al., 2010), a very important signal during salt stress. In line with this results leaf nitrate reductase activ-ity, also related to enzymatic nitric oxide production (Yamasaki and Sakihama, 2000) and assimilatory nitrogen metabolism, was affected by both NaCl and HAs (Fig 1).Conclusions Tomato plants previously treated with HAs and then challenged with NaCI exhibited increased sodium tolerance, by a mechanism related to auxin-like effects on proton pumps, holding nitrate reduc-tase activity and photosynthesis in the same level as unstressed plants. We cannot exclude the possible interaction of HAs with mineral nutrients during NaCl stress. It remains to be elucidated whether mitigat-ing mechanisms are due auxin-like molecules of HAs VWUXFWXUH RU PRGL¿FDWLRQ RI HQGRJHQRXV SODQW KRU-mone balance by HAs. Despite the promising results presented here, the role of HAs as an addictive for fer-tilizers need more research mainly regarding its posi-WLYHHIIHFWVLQ¿HOGFRQGLWLRQV.H\ZRUGVRUJDQLFPDWWHUSKRWRV\QWKHVLVELRVWLP-ulant, proton pumps.Acknowledgements:HJUDWHIXOO\DFNQRZOHGJHWKH¿QDQFLDOVXSSRUWRIthe National Counsel of Technological and Scien-WL¿F'HYHORSPHQW&13TReferencesMora, V., M. Olaetxea, E. Bacaicoa, R. Baigorri, M. Fuentes, A. M. Zamarreño, and J. M. Garcia-Mina. “Abiotic Stress Tolerance in Plants: Exploring the Role of Nitric Oxide and Humic Substances.” In Ni-tric Oxide in Plants: Metabolism and Role in Stress Physiology, pp. 243-264. Springer International Pu-blishing, 2014. Baigorri, R., Urrutia, O., Erro, J., Mandado, M., Pé-rez-Juste, I., & Garcia-Mina, J. M. (2013). Structural Characterization of Anion–Calcium–Humate Com-plexes in Phosphate-based Fertilizers. ChemSu-sChem, 6(7), 1245-1251.Binzel, M. L. (1995). NaCl-induced accumulation of tonoplast and plasma membrane H+-ATPase mes-sage in tomato. Physiologia Plantarum, 94(4), 722-728.Bot A, Benites J (2005) The importance of soil or-JDQLFPDWWHU.H\WRGURXJKWUHVLVWDQWVRLODQGVXV-tained food production. FAO, Rome.Dunlap, J. R., & Binzel, M. L. (1996). NaCI reduces indole-3-acetic acid levels in the roots of tomato plants independent of stress-induced abscisic acid. Plant physiology, 112(1), 379-384.FAO, 2007. Extent and Causes of Salt-affected Soils in Participating Countries. AGL: Global Net-work on Integrated Soil Management for Sustain-able use of Salt-affected Soils. <http://www.fao.org/DJDJODJOOVSXVKWRSLFKWP!Jannin, L., Arkoun, M., Ourry, A., Laîné, P., Goux, D., Garnica, M., ... & Etienne, P. (2012). Microarray analysis of humic acid effects on Brassica napus growth: Involvement of N, C and S metabolisms. Plant and soil, 359(1-2), 297-319.Liu, C., & Cooper, R. J. (2002). Humic acid appli-cation does not improve salt tolerance of hydro-ponically grown creeping bentgrass. Journal of the American Society for Horticultural Science, 127(2), 219-223.Yamasaki, H., & Sakihama, Y. (2000). Simultane-ous production of nitric oxide and peroxynitrite by Posters | Section  8 | Organic based fertilizers16th World Fertilizer Congress of CIEC246plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species. Febs Letters, 468(1), 89-92.Figure 1. Root plasma membrane H+-ATPase and tonoplast H+-PPase activities, leaf nitrate reductase activity, root malondialdehyde (MDA) content and root membrane integrityZandonadi, D. B., Santos, M. P., Dobbss, L. B., Oli-vares, F. L., Canellas, L. P., Binzel, M. L., ... & Fa-çanha, A. R. (2010). Nitric oxide mediates humic acids-induced root development and plasma mem-brane H+-ATPase activation. Planta, 231(5), 1025-1036.Figure 2. Stomatic condutance, photosynthetic rate, transpiration rate and chlorophyll a content of tomato plants

Mitigation of salt stress of tomato using pre-treatment with humic acids.

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