Assessing Changes in Root Architecture, Developmental Timing, Transcriptional and Hormonal Profiles in Rice Co-Cultivated with Azolla filiculoides

doi:10.1016/j.rsci.2025.03.004

Abstract

Abstract:

Strategies for increasing rice yield are needed to keep pace with the expected global population growth and sustainably address the challenges posed by climate change. In Southeast Asian countries, rice farming benefits from the use of Azolla spp. for nitrogen supply. By virtue of their symbiosis with the nitrogen-fixing cyanobacterium Trichormus azollae, Azolla spp. are ferns that release nitrogen into the environment upon biomass decomposition. However, whether and to what extent actively growing Azolla plants influence the development of co-cultivated rice seedlings remains unclear. To address this, rice (Oryza sativa L. var. Kitaake) seedlings were co-cultivated hydroponically with Azolla filiculoides for up to two months. Morphological changes in rice roots and aerial organs were assessed alongside nitric oxide assays in rice roots, root transcriptomics, and targeted hormonomics of rice roots, leaves, and growth media. Here, we showed that co-cultivation with actively growing A. filiculoides alters rice root architecture by inducing a nitric oxide boost and accelerates leaf and tiller differentiation and proliferation. Overall, this study provides an in-depth analysis of the morphogenetic effects of co-cultivated A. filiculoides on rice during early vegetative growth. It also paves the way for studies assessing whether A. filiculoides co-cultivation primes rice plants to better withstand abiotic and biotic stresses.

Key words: Azolla, hormone, liquid chromatography-mass spectrometry, Oryza sativa, nitric oxide, plant architecture, root apparatus, transcriptome, Trichormus (Anabaena) azollae

Sara Cannavò, Chiara Paleni, Alma Costarelli, Maria Cristina Valeri, Martina Cerri, Antonietta Saccomanno, Veronica Gregis, Graziella Chini Zittelli, Petre I. Dobrev, Lara Reale, Martin M. Kater, Francesco Paolocci. Assessing Changes in Root Architecture, Developmental Timing, Transcriptional and Hormonal Profiles in Rice Co-Cultivated with Azolla filiculoides[J]. Rice Science, 2025, 32(3): 426-444.

Figures/Tables 10

Fig. 1. Effect of Azolla filiculoides on the number (A) and length (B) of adventitious roots (ARs) in rice at 15, 30, and 60 d after transplanting (DAT), and on the number of rice ARs with (+LR) and without (-LR) lateral roots along with +LR/-LR ratio at 15 (C) and 30 DAT (D). Data represent Mean ± SE of control rice (R) and rice grown with A. filiculoides (R + Af). In A, n = 6 and 7 for R and R + Af at 15 DAT, respectively; n = 254 and 333 for R and R + Af at 30 DAT, respectively; n = 853 and 1 711 for R and R + Af at 60 DAT, respectively. In B, n = 144 and 187 for R and R + Af at 15 DAT, respectively; n = 18 for R and R + Af at 30 DAT; n = 7 and 9 for R and R + Af at 60 DAT, respectively. In C and D, n = 10 and 11 for R and R + Af at 15 and 30 DAT, respectively. *, P < 0.05; ***, P < 0.001.

Fig. 2. Effect of Azolla filiculoides on relative amount of nitric oxide in adventitious roots (ARs) of rice at 15 and 30 d after transplanting (DAT). A, Nitric oxide levels assayed using fluorescence analysis after staining with 4,5-diaminofluorescein diacetate (DAF-2DA) probe. CTCF, Corrected total cell fluorescence. Data represent Mean ± SE of control rice (R) and rice grown with A. filiculoides (R + Af). n = 23 and 22 for R and R + Af at 15 DAT, respectively; n = 15 for R and R + Af at 30 DAT. ***, P < 0.001. B, Fresh sections of adventitious apex at 15 DAT stained with DAF-2DA probe. Pictures were taken under an epifluorescence light microscopy (excitation 495 nm; emission 515 nm; long-pass filter of 515 nm). Scale bars, 200 μm.

Fig. 3. Effect of Azolla filiculoides on the number of leaves (A), plant height (B), number of tillers (C), and root/shoot ratio of fresh weights (D) over two months. DAT, Days after transplanting. Data represent Mean ± SE of control rice (R) and rice grown with A. filiculoides (R + Af). In A‒C, n = 12 for R and R + Af at 0, 7, 14, 21, and 28 DAT; n = 11 and 12 for R and R + Af at 35 and 42 DAT, respectively; n = 8 and 9 for R and R + Af at 49, 56, and 63 DAT, respectively; In D, n = 6 and 7 for R and R + Af at 15 DAT, respectively; n = 20 for R and R + Af at 30 DAT; n = 121 and 15 for R and R + Af at 60 DAT, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 4. Principal component analysis (PCA, A) and differentially expressed gene (DEG) count at 15 d after transplanting (B). R, Rice, used as a control; R + Af, Rice co-cultivated with Azolla filiculoides.

Fig. 5. Twenty most enriched biological process (A) and molecular function (B) terms among differentially expressed genes in rice roots at 15 d after transplanting. FDR, False discovery rate.

Fig. 6. Schematic representation of iron acquisition systems in rice roots. Under Strategy I, the ferric chelate complex is reduced to ferrous ion by ferric chelate reductase (FRO). H+ released by the proton pump and phenolics released by phenolics efflux transporter (PEZ) acidify the medium to convert ferric ion to ferrous ion. Iron transporters (IRTs) transport the ferrous ion into the root cells. Vacuolar iron transporters (VITs) are responsible for transportation and accumulation of iron within the vacuole while ferritins (FERs) are the proteins accommodating excess iron within the cells. Under Strategy II, mugineic acid (MA) phytosiderophores, produced by the sequential activity of nicotianamine synthase (NAS), nicotianamine aminotransferase (NAAT), and deoxymugineic acid synthase (DMAS). Deoxymugineic acid efflux transporter 1 (TOM1) transporter mediates the DMA secretion through the plasma membrane. Chelated Fe(III) complex is transported across the membrane into the root cell by Yellow Stripe-Like (YSL) transporters. Green and red arrows refer to transporters/enzymes whose mRNA are increased and decreased, respectively, in the R + Af (rice grown with Azolla filiculoides) vs R (rice) treatment. For the differential expression of regulatory genes refer to Table S2. The picture was redrawn from Kobayashi et al (2014) and Kar and Panda (2020).

Table 1. Nitrate oxide-related differentially expressed genes at 15 d after transplanting.

Accession number	Symbol	log₂(fold change)	Adjusted P-value	Description
Os02g0770800	NR2/NIA1/NIA2	5.373	8.4E-05	NADH/NADPH-dependent nitrate reductase
Os10g0554200	NRT1.1B/NPF6.5	3.049	3.9E-04	Nitrate transporter 1.1B
Os10g0169900	NRT	4.308	1.6E-04	Nitrate and chloride transporter

Fig. 7. Gene expression profiles of selected differentially expressed genes at 15 d after transplanting (DAT) as resulted from qRT-PCR analysis at 0, 5, 9 12, and 15 DAT. R, Rice, used as a control; R + Af, Rice co-cultivated with Azolla filiculoides. Asterisks indicate significant differences between the two treatments at a given time pint (t-test, P < 0.05), while lowercase letters above bars indicate significant differences between time points of the same treatment (Analysis of variance, P < 0.05; Tukey’s HSD, P < 0.05).

Fig. 8. Differential levels of hormonal compounds in roots of rice grown with and without Azolla filiculoides at 15 d after transplanting. Unpaired two samples t-test for all five compounds except IAA-Glu, for which the unpaired two samples of Wilcoxon test was performed. For all five compounds: P < 0.01 and false discovery rate cut-off ≤ 0.05. R, Rice, used as a control; R + Af, Rice co-cultivated with Azolla filiculoides; iP7G, Isopentenyl adenine-7-glucoside; ABA, Abscisic acid; DPA, Dihydrophaseic acid; IAA-Glu, Indole-3-acetic acid-glutamate; SinAc, Sinapic acid.

Fig. 9. Accumulation profiles at 0 and 15 d after transplanting of bioactive hormones in rice roots (A) and leaves (B) from R + Af and R treatments. Asterisks indicate significant differences between two treatments at a given time pint (t-test, P < 0.05), while lowercase letters indicate significant differences between time points of the same treatment (Analysis of variance, P < 0.05; Tukey’s HSD, P < 0.05). R, Rice, used as a control; R + Af, Rice co-cultivated with Azolla filiculoides; ABA, Abscisic acid; IAA, Indole-3-acetic acid; SA, Salicylic acid; JA, Jasmonic acid; cZ, cis-Zeatin; DZ, Dihydrozeatin; tZ, trans-Zeatin; iP, Isopentenyl adenine.

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