Humic Substances Isolated from Recycled Biomass Trigger Jasmonic Acid Biosynthesis and Signalling

Abstract

Intensive agriculture maintains high crop yields through chemical inputs, which are well known for their adverse effects on environmental quality and human health. Innovative technologies are required to reduce the risk generated by the extensive and harmful use of pesticides. The plant biostimulants made from humic substances isolated from recyclable biomass offer an alternative approach to address the need for replacing conventional agrochemicals without compromising the crop yield. The stimulatory effects of humic substances are commonly associated with plant hormones, particularly auxins. However, jasmonic acid (JA) is crucial metabolite in mediating the defence responses and governing plant growth and development. This work aimed to evaluate the changes in the biosynthesis and signalling pathway of JA in tomato seedlings treated with humic acids (HA) isolated from vermicompost. We use the tomato model system cultivar Micro-Tom (MT) harbouring a reporter gene fused to a synthetic promoter that responds to jasmonic acid (JERE::GUS). The transcript levels of genes involved in JA generation and activity were also determined using qRT-PCR. The application of HA promoted plant growth and altered the JA status, as revealed by both GUS and qRT-PCR assays. Both JA enzymatic synthesis (LOX, OPR3) and JA signalling genes (JAZ and JAR) were found in higher transcription levels in plants treated with HA. In addition, ethylene (ETR4) and auxin (ARF6) signalling components were positively modulated by HA, revealing a hormonal cross-talk. Our results prove that the plant defence system linked to JA can be emulated by HA application without growth inhibition.

Keywords: biotic stress; eco-compatible chemicals; hormonal effect.

Figure 1

Spectrum of IR-TF (a) and CP/MAS 13C NMR (b) of humic acids isolated from vermicompost.

Figure 2

(A) Phenotypes of 30-day-old MT tomatoes seedlings exposed to water as a control, 1 mM MeJA, 4 and 8 mM C HA (humic acids). Fresh weight of roots (B) and shoots (C) treated with 1 mM MeJA, 4 and 8 mM C HA. Data represent the mean and bars standard deviation (n = 10). Means followed by different letters are significantly different by the LSD test (p < 0.05).

Figure 3

Leaves of Micro-Tom (MT) with gene reporter JERE: GUS were used to perform the GUS staining assays: (A) control, (B) 1 mM MeJ, (C) HA 4 mM C L⁻¹, (D) HA 8 mM C L⁻¹, (E) HA 16 mM C L⁻¹, (F) HA 32 mM C L⁻¹.

Figure 4

Gene expression linked to jasmonate biosynthesis. phospholipase1 (PLDa1), lipoxygenase (LOX2), alene oxide cyclase (AOC), 12-oxo-phytodienoic acid (OPDA) reductase3 (OPR3) genes in MT tomatoes treated with 1 mM of methyl jasmonate (MeJA) and 4 and 8 mM C of humic acids (HA) isolated from vermicompost. Total RNA was extracted from leaves and subjected to real-time qPCR analysis. Data represent the mean of three independent samples with SD. * significant difference at p < 0.05 by t test. The data are expressed concerning control treatment considered = 0.

Figure 5

Gene expression linked to jasmonate signalling. bHLH transcription Factor (MYC2) jasmonate zim domain (JAZ) and jasmonic acid-amino acid synthetase (JAR) in tomato MT exogenous treated with 1 mM methyl jasmonate (MeJA) and 4 and 8 mM C of humic acids (HA) isolated from vermicompost. Total RNA was extracted from leaves and subjected to real-time qPCR analysis. Data represent the mean of three independent samples with SD. * significant difference at p < 0.05 by t test. The data are expressed concerning control treatment considered = 0.

Figure 6

Gene expression linked to herbivory (cellulose synthase-CEVI57), ethylene-jasmonate cross-talk (ethylene response sensor-ETR4) and jasmonate-auxin cross-talk (auxins response factors, ARF6 and ARF8) in tomato MT exogenous treated with 1 mM methyl jasmonate (MeJA) and 4 and 8 mM C of humic acids (HA) isolated from vermicompost. Total RNA was extracted from leaves and subjected to real-time qPCR analysis. Data represent the mean of three independent samples with SD. * significant difference at p < 0.05 by t test. The data are expressed concerning control treatment considered = 0.

Figure 7

Effect of methyl jasmonate (MeJA) and 4 and 8 mM C of humic acids (HA) isolated from vermicompost on glandular trichome density on MT tomato leaves. (A) mean density of type VI glandular trichome on the adaxial (upper) leaf surface and (B) abaxial (lower) l leaf surface. N = 10 plants per treatment, one leaf examined per plant. Densities are calculated from counts of trichomes on two leaf disks per leaf. Data represent the mean and bar standard deviation. Means followed by different letters are significantly different from the LSD test (p < 0.05).