Multiple functions of glomalin released by arbuscular mycorrhizal fungi are well-recognized, whereas the role of exogenous glomalins including easily extractable glomalin-related soil protein (EE-GRSP) and difficultly extractable glomalin-related soil protein (DE-GRSP) is unexplored for plant responses. Our study was carried out to assess the effects of exogenous EE-GRSP and DE-GRSP at varying strengths on plant growth and chlorophyll concentration of trifoliate orange (Poncirus trifoliata) seedlings, along with changes in root nutrient acquisition, auxin content, auxin-related enzyme and transporter protein gene expression, and element contents of purified GRSP. Sixteen weeks later, exogenous GRSP displayed differential effects on plant growth (height, stem diameter, leaf number, and biomass production): the increase by EE-GRSP and the decrease by DE-GRSP. The best positive effect on plant growth occurred at exogenous EE-GRSP at ½ strength. Similarly, the GRSP application also differently affected total chlorophyll content, root morphology (total length, surface area, and volume), and root N, P, and K content: positive effect by EE-GRSP and negative effect by DE-GRSP. Exogenous EE-GRSP accumulated more indoleacetic acid (IAA) in roots, which was associated with the upregulated expression of root auxin synthetic enzyme genes (PtTAA1, PtYUC3, and PtYUC4) and auxin influx transporter protein genes (PtLAX1, PtLAX2, and PtLAX3). On the other hand, exogenous DE-GRSP inhibited root IAA and indolebutyric acid (IBA) content, associated with the downregulated expression of root PtTAA1, PtLAX1, and PtLAX3. Root IAA positively correlated with root PtTAA1, PtYUC3, PtYUC4, PtLAX1, and PtLAX3 expression. Purified EE-GRSP and DE-GRSP showed similar element composition but varied in part element (C, O, P, Ca, Cu, Mn, Zn, Fe, and Mo) concentration. It concluded that exogenous GRSP triggered differential effects on growth response, and the effect was associated with the element content of pure GRSP and the change in auxins and root morphology. EE-GRSP displays a promise as a plant growth biostimulant in citriculture.

Botany and Microbiology Department College of Science King Saud University Riyadh Saudi Arabia

College of Horticulture and Gardening Yangtze University Jingzhou China

Department of Chemistry Faculty of Science University of Hradec Kralove Hradec Kralove Czechia

ICAR Central Citrus Research Institute Nagpur India

Plant Production Department College of Food and Agricultural Sciences King Saud University Riyadh Saudi Arabia

See more in PubMed

Arnon D. I. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15. 10.1104/pp.24.1.1 PubMed DOI PMC

Bradford M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 10.1016/0003-2697(76)90527-3 PubMed DOI

Brumos J., Alonso J. M., Stepanova A. N. (2014). Genetic aspects of auxin biosynthesis and its regulation. Physiol. Plant. 151, 3–12. 10.1111/ppl.12098 PubMed DOI

Chen D., Wang W. A., Yue Q. Q., Zhao Q. (2016). Research progress of plant auxin as a regulator of cold stress response. J. Plant Physiol. 7, 989–997. 10.13592/j.cnki.ppj.2016.0042 DOI

Chi G. G., Srivastava A. K., Wu Q. S. (2018). Exogenous easily extractable glomalin-related soil protein improves drought tolerance of trifoliate orange. Arch. Agron. Soil Sci. 64, 1341–1350. 10.1080/03650340.2018.1432854 DOI

Delbarre A., Muller P., Guern I. J. (1996). Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells. Planta 198, 532–541. 10.1007/BF00262639 PubMed DOI

Dobrev P. I., Kamínek M. (2002). Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J. Chromatogr. A 950, 21–29. 10.1016/S0021-9673(02)00024-9 PubMed DOI

Dong S. S., Dou S., Lin C. M., Li L. B., Tan C. (2016). Decomposition rate of corn straw in soil and its effects on soil humus composition. J. Jilin Agric. Univ. 38, 579–586. 10.13327/j.jjlau.2016.3373 DOI

Duan D. C. (2014). Regulation Mechanism of Humic Acid on the Pb Bioavailability and Toxicity to Tea Plant (Cameilia sinensis L.). [Doctor's thesis]. [Hangzhou]: Zhejiang University.

Gadkar V., Rillig M. C. (2006). The arbuscular mycorrhizal fungal protein glomalin is a putative homolog of heat shock protein 60. FEMS Microbiol. Lett. 263, 93–101. 10.1111/j.1574-6968.2006.00412.x PubMed DOI

Gao W. Q., Wang P., Wu Q. S. (2019). Functions and application of glomalin-related soil proteins: a review. Sains Malays. 48, 111–119. 10.17576/jsm-2019-4801-13 DOI

Gillespie A. W., Farrell R. E., Walley F. L., Ross A. R. S., Leinweber P., Eckhardt K. U. (2011). Glomalin-related soil protein contains non-mycorrhizal related heat-stable proteins, lipids and humic materials. Soli Biol. Biochem. 43, 766–777. 10.1016/j.soilbio.2010.12.010 PubMed DOI

He J. D., Chi G. G., Zou Y. N., Shu B., Wu Q. S., Srivastava A. K., et al. . (2020). Contribution of glomalin-related soil proteins to soil organic carbon in trifoliate orange. Agric. Ecosyst. Environ,. Appl. Soil Ecol. 154:103592. 10.1016/j.apsoil.2020.103592 DOI

Huang G. M., Srivastava A. K., Zou Y. N., Wu Q. S., Kuča K. (2021a). Exploring arbuscular mycorrhizal symbiosis in wetland plants with a focus on human impacts. Symbiosis. 10.1007/s13199-021-00770-8. DOI

Huang Y., Xu Y. T., Jiang X. L., Yu H. W., Jia H. H., Tan C. M., et al. . (2021b). Genome of a citrus rootstock and global DNA demethylation caused by heterografting. Hortic. Res. 8:69. 10.1038/s41438-021-00505-2 PubMed DOI PMC

Koide R. T., Peoples M. S. (2013). Behavior of Bradford-reactive substances is consistent with predictions for glomalin. Agric. Ecosyst. Environ. Appl. Soil Ecol. 63, 8–14. 10.1016/j.apsoil.2012.09.015 DOI

Li L., Ljung K., Breton G., Schmitz R. J., Pruneda-Paz J., Cowing-Zitron C., et al. . (2012). Linking photoreceptor excitation to changes in plant architecture. Gene Dev. 26, 785–790. 10.1101/gad.187849.112 PubMed DOI PMC

Liu C. Y., Wang P., Zhang D. J., Zou Y. N., Kuča K., Wu Q. S. (2018a). Mycorrhiza-induced change in root hair growth is associated with IAA accumulation and expression of EXPs in trifoliate orange under two P levels. Sci. Hortic. 234, 227–235. 10.1016/j.scienta.2018.02.052 DOI

Liu C. Y., Zhang F., Zhang D. J., Srivastava A. K., Wu Q. S., Zou Y. N. (2018b). Mycorrhiza stimulates root-hair growth and IAA synthesis and transport in trifoliate orange under drought stress. Sci. Rep. 8:1978. 10.1038/s41598-018-20456-4 PubMed DOI PMC

Livak K. J., Schmittgen T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT. Methods 25, 402–408. 10.1006/meth.2001.1262 PubMed DOI

Lovelock C. E., Wright S. F., Clark D. A., Ruess R. W. (2004). Soil stocks of glomalin produced by arbuscular mycorrhizal fungi across a tropical rain forest landscape. J. Ecol. 92, 278–287. 10.1111/j.0022-0477.2004.00855.x DOI

Meng L. L., He J. D., Zou Y. N., Wu Q. S., Kuča K. (2020). Mycorrhiza-released glomalin-related soil protein fractions contribute to soil total nitrogen in trifoliate orange. Plant Soil Environ. 66, 183–189. 10.17221/100/2020-PSE DOI

Mora V., Bacaicoa E., Zamarreño A. M., Aguirre E., Garcia-Mina J. M. (2012). Action of humic acid on promotion of cucumber shoot growth involves nitrate-related changes associated with the root-to-shoot distribution of cytokinins, polyamines and mineral nutrients. J. Plant Physiol. 167, 633–642. 10.1016/j.jplph.2009.11.018 PubMed DOI

Nichols K. A. (2008). Indirect contributions of AM fungi and soil aggregation to plant growth and protection, in Sustainable Agriculture and Forestry, Siddiqui Z. A.. (Berlin: Springer; ), 177–194. 10.1007/978-1-4020-8770-7_7 DOI

Peret B., Swarup K., Ferguson A., Seth M., Yang Y. D., Dhondt S., et al. . (2012). AUX/LAX genes encode a family of auxin influx transporters that perform distinct functions during Arabidopsis development. Plant Cell 24, 2874–2885. 10.1105/tpc.112.097766 PubMed DOI PMC

Rillig M. C. (2004). Arbuscular mycorrhizae, glomalin and soil aggregation. Can. J. Soil Sci. 84, 355–363. 10.4141/S04-003 DOI

Rillig M. C., Mummey D. L. (2010). Mycorrhizas and soil structure. New Phytol. 171, 41–53. 10.1111/j.1469-8137.2006.01750.x PubMed DOI

Rillig M. C., Wright S. F., Nichols K. A., Schmidt W. F., Torn M. S. (2001). Unusually large contribution of arbuscular mycorrhizal fungi to soil organic matter pools in tropical forest soils. Plant Soil 233, 167–177. 10.1023/A:1010364221169 DOI

Schindler F. V., Mercer E. J., Rice J. A. (2007). Chemical characteristics of glomalin-related soil protein (GRSP) extracted from soils of varying organic matter content. Soil Biol. Biochem. 39, 320–329. 10.1016/j.soilbio.2006.08.017 DOI

Spohn M., Giani L. (2010). Water-stable aggregates, glomalin-related soil protein, and carbohydrates in a chronosequence of sandy hydromorphic soils. Soil Biol. Biochem. 42, 1505–1511. 10.1016/j.soilbio.2010.05.015 DOI

Sun J. Q., Qi L. L., Li Y. N., Chu J. F., Li C. Y. (2012). PIF4-mediated activation of YUCCA8 expression integrates temperature into the auxin pathway in regulating Arabidopsis hypocotyl growth. PLoS Genet. 8:e1002594. 10.1371/journal.pgen.1002594 PubMed DOI PMC

Swarup K., Benkova E., Swarup R., Casimiro I., Peret B., Yang Y. D., et al. . (2008). The auxin influx carrier LAX3 promotes lateral root emergence. Nat. Cell Biol. 10, 946–954. 10.1038/ncb1754 PubMed DOI

Wang S., Wu Q. S., He X. H. (2015). Exogenous easily extractable glomalin-related soil protein promotes soil aggregation, relevant soil enzyme activities and plant growth in trifoliate orange. Plant Soil Environ. 61, 66–71. 10.17221/833/2014-PSE DOI

Wang Y., Wang M., Li Y., Wu A., Huang J. (2018). Effects of arbuscular mycorrhizal fungi on growth and nitrogen uptake of Chrysanthemum morifolium under salt stress. PLoS ONE 7:12181. 10.1371/journal.pone.0196408 PubMed DOI PMC

Wu Q. S., Cao M. Q., Zou Y. N., He X. H. (2014). Direct and indirect effects of glomalin, mycorrhizal hyphae, and roots on aggregate stability in rhizosphere of trifoliate orange. Sci. Rep. 4:5823. 10.1038/srep05823 PubMed DOI PMC

Wu Q. S., Li Y., Zou Y. N., He X. H. (2015a). Arbuscular mycorrhiza mediates glomalin-related soil protein production and soil enzyme activities in the rhizosphere of trifoliate orange grown under different P levels. Mycorrhiza 25, 121–130. 10.1007/s00572-014-0594-3 PubMed DOI

Wu Q. S., Srivastava A. K., Wang S., Zeng J. X. (2015b). Exogenous application of EE-GRSP and changes in citrus rhizosphere properties. Ind. J. Agric. Sci. 85, 58–62.

Xu D. B., Wang Q. J., Wu Y. C., Yu G. H., Shen Q. R., Huang Q. W. (2012). Humic-like substances from different compost extracts could significantly promote cucumber growth. Pedosphere 22, 815–824. 10.1016/S1002-0160(12)60067-8 DOI

Yoshihiro M., Keiichirou N. (2012). The pathway of auxin biosynthesis in plants. J. Exp. Bot. 8, 2853–2872. 10.1093/jxb/ers091 PubMed DOI

Zhang B. (2009). Effects of MAG2 Gene on the Germination and the Growth of Seedling in Arabidopsis thaliana Under the Stress Condition. [Master's thesis]. [Lanzhou]: Lanzhou University.

Zhang Z. H., Wang Q., Wang H., Nie S. M., Liang Z. W. (2017a). Effects of soil salinity on the content, composition, and ion binding capacity of glomalin-related soil protein. Sci. Total Environ. 581, 657–665. 10.1016/j.scitotenv.2016.12.176 PubMed DOI

Zhang Z. L., Wang W. J., Wang Q., Wu Y., Wang H. M., Pei Z. X. (2017b). Glomalin amount and compositional variation, and their associations with soil properties in farmland, northeastern China. J. Plant Nutr. Soil Sci. 180, 563–575. 10.1002/jpln.201600579 DOI

Zhao R. X., Guo W., Bi N., Guo J. Y., Wang L. X., Zhao J., et al. . (2015). Arbuscular mycorrhizal fungi affect the growth, nutrient uptake and water status of maize (Zea mays L.) grown in two types of coal mine spoils under drought stress. Agric. Ecosyst. Environ. Appl. Soil Ecol. 88, 41–49. 10.1016/j.apsoil.2014.11.016 DOI

Zheng Y., Zhou A. P., Liu Y. K., He C. Z. (2013). The polar transport and regulatory mechanism of auxin in plant. J. Yunnan Agric. Univ. 6, 122–128. 10.3969/j.issn.1004-390X(n).2013.06.020 PubMed DOI

Zou Y. N., Srivastava A. K., Wu Q. S. (2015). Glomalin: a potential soil conditioner for perennial fruits. Int. J. Agric. Biol. 18, 293–297. 10.17957/IJAB/15.0085 DOI

Zou Y. N., Srivastava A. K., Wu Q. S., Huang Y. M. (2014). Glomalin-related soil protein and water relations in mycorrhizal citrus (Citrus tangerina) during soil water deficit. Arch. Agron. Soil Sci. 60, 1103–1114. 10.1080/03650340.2013.867950 DOI

Serendipita indica promotes P acquisition and growth in tea seedlings under P deficit conditions by increasing cytokinins and indoleacetic acid and phosphate transporter gene expression