Facilitating Phloem-Mediated Iron Transport Can Improve the Adaptation of Rice Seedlings to Iron Deficiency Stress

Abstract

Iron (Fe) is essential for normal plant growth and development. In rice, Fe deficiency leads to stunted growth, leaf chlorosis, reduced photosynthetic capacity, and ultimately, yield loss. Most studies have focused on investigating the mechanisms of Fe deficiency responses in rice roots; however, the effects of shoot Fe redistribution on Fe deficiency response remain poorly understood. Phloem transport plays a vital role in distributing Fe to new tissues. To investigate the effects of enhanced phloem-mediated Fe transport on rice adaptability to iron deficiency, we subjected transgenic lines with higher phloem Fe efflux rates and wild-type (WT) plants to Fe-deficient conditions. The growth, leaf photosynthetic rate, and Fe content of transgenic and WT seedlings under different Fe concentrations were compared. The results showed that the transgenic lines exhibited elevated shoot length, root length, shoot dry weight, leaf chlorophyll content, and net photosynthetic rates under Fe-deficient conditions. Under both Fe-sufficient and Fe-deficient conditions, the transgenic lines had significantly higher Fe content, Fe accumulation, and phloem Fe efflux rates than the WT. RNA sequencing (RNA-seq) analysis revealed that enhanced Fe transport via phloem resulted in improved Fe availability through the sequestration of Fe ions and vacuolar transport pathways in the shoots. It also upregulated the EARLY LESION LEAF 1 (ELL1) expression and modulated the sucrose synthase activity, thereby promoting chlorophyll synthesis and leaf photosynthesis. Additionally, enhanced Fe transport influenced the gibberellin (GA) catabolism and plant hormone signal transduction in the roots, reducing the GA content and modulating the cytokinin (CTK), jasmonic acid (JA), and ethylene (ETH) signaling to induce Fe deficiency response and promote Fe uptake. These findings demonstrate that phloem-mediated Fe transport participated in Fe deficiency response, and enhancing this improved the adaptability of rice seedlings to low Fe conditions. In specific, rice seedlings with a high capacity for phloem-mediated Fe transport exhibited a strong iron uptake, translocation, and remobilization capacity, thereby maintaining normal growth and development and successfully adapting to the low-Fe environment.

Keywords: Iron Deficiency; Iron transport; Phloem; Plant hormone signal transduction; Rice (Oryza Sativa L.).

Fig. 1

Seedling growth of wild-type (WT) and transgenic pOsSUT1::OsYSL9 line 3 (SY-3) and pOsSUT1::OsIRT1 line 4 (SI-4) rice at 7 days after treatment (DAT) with 10 and 40 µM iron (Fe). A Photograph of seedlings. Scale bar = 1 cm. Shoot (B) and root (C) lengths (n = 10 replicates). Dry weight of the shoots (D) and roots (E) (n = 3 replicates). Data are presented as means ± SD. Different letters indicate significance based on Duncan’s test (P < 0.05)

Fig. 2

Leaf chlorophyll contents and photosynthetic rates in wild-type (WT) and transgenic SY-3 and SI-4 rice at 7 days after treatment (DAT) with 10 and 40 µM iron (Fe). A Photograph of leaf color. SPAD values (B) and total chlorophyll contents (C) of the 4th leaves. SPAD values (D) and total chlorophyll contents (E) of the 3rd leaves. F Net photosynthetic rate of the 3rd leaves. Data in B, D and F represent the means ± SD of five replicates. Data in C and E represent the means ± SD of four replicates. Different letters indicate significance based on Duncan’s test (P < 0.05)

Fig. 3

Iron status in wild-type (WT) and transgenic SY-3 and SI-4 rice at 7 days after treatment (DAT) with 10 and 40 µM iron (Fe). Fe content in the shoots (A) and roots (B). C Fe efflux rate of the leaf phloem at 3 DAT (n = 4 replicates). Fe accumulation in the shoots (D), roots (E), and whole plants (F). Data in A, B, D, E, and F represent the means ± SD of three replicates. Different letters indicate significance based on Duncan’s test (P < 0.05)

Fig. 4

RNA sequencing analysis of the leaves (L) and roots (R) from wild-type (WT) and transgenic SY-3 and SI-4 rice after treatment with 10 and 40 µM iron (Fe). A Diagram of the experimental setup. B Principal component analysis of identified genes. C Number of differentially expressed genes (DEGs) in the leaves and roots per treatment. Up, upregulated; Down, downregulated. Venn diagrams of DEGs in the leaves (D) and roots (E)

Fig. 5

Functional analysis of leaf-specific differentially expressed genes (DEGs) in response to iron deficiency. Hierarchical clustering (A) and KEGG (B), GO-MF (C), and GO-BP (D) enrichment analyses of 149 DEGs common to the wild-type (WT) and transgenic SY-3 and SI-4. Hierarchical clustering (E) and KEGG (F), GO-MF (G), and GO-BP (H) enrichment analyses of 260 DEGs unique to SY-3 and SI-4

Fig. 6

Key leaf-specific differentially expressed genes (DEGs) associated with iron deficiency response in wild-type (WT) and transgenic SY-3 and SI-4 rice. A Heat map of key DEGs associated with ‘plant hormone signal transduction’, ‘starch and sucrose metabolism’, ‘iron ion homeostasis’, ‘ion transmembrane transporter activity’, and ‘iron ion binding’. B Heat map of key DEGs unique to SY-3 and SI-4 and associated with ‘starch and sucrose metabolism’, ‘plant hormone signal transduction’, ‘iron ion binding’, ‘sequestering of iron ion’, and ‘ubiquitin protein ligase activity’. Data represent the means of three replicates

Fig. 7

Expression validation of iron deficiency response genes in the leaves of wild-type (WT) and transgenic SY-3 and SI-4 rice. Relative expression of OsVIT2 (A), OsFER1 (B), OsFER2 (C), OsSUS1 (D), OsTPS7 (E), and ELL1 (F). Data represent the means ± SD of three replicates. Different letters indicate significance based on Duncan’s test (P < 0.05)

Fig. 8

Functional analysis of root-specific differentially expressed genes (DEGs) in response to iron deficiency. Hierarchical clustering (A) and KEGG (B), GO-MF (C), and GO-BP (D) enrichment analyses of 282 DEGs common to the wild-type (WT) and transgenic SY-3 and SI-4. Hierarchical clustering (E) and KEGG (F), GO-MF (G), and GO-BP (H) enrichment analyses of 934 DEGs unique to SY-3 and SI-4

Fig. 9

Key root-specific differentially expressed genes (DEGs) associated with iron deficiency response in the wild-type (WT) and transgenic SY-3 and SI-4 rice. A Heat map of key DEGs associated with ‘transmembrane transporter activity’, ‘nicotinamide synthase activity’, and ‘iron ion binding’. B Heat map of key DEGs unique to SY-3 and SI-4 and associated with ‘transmembrane transporter activity’, ‘nicotinamide synthase activity’, and ‘iron ion binding’. Data represent the means of three replicates

Fig. 10

Expression validation of iron deficiency response genes in the roots of wild-type (WT) and transgenic SY-3 and SI-4 rice. Relative expression of OsGA2ox8 (A), OsRR6 (B), OsCTR2 (C), OsEBF1 (D), OsJAZ8 (E), and OsABF1 (F). Data represent the means ± SD of three replicates. Different letters indicate significance based on Duncan’s test (P < 0.05)

Fig. 11

Hormone contents in wild-type (WT) and transgenic SY-3 and SI-4 rice roots after treatment with 10 and 40 µM iron: GA₄ (A) and tZ (B). Data represent the means ± SD of three replicates. Different letters indicate significance based on Duncan’s test (P < 0.05)

Fig. 12

Schematic diagram showing the potential mechanism to improve iron (Fe) deficiency tolerance in rice thorough enhanced phloem-mediated Fe transport. Under Fe-deficient conditions, Fe storage in transgenic SY and SI leaves is reduced by the downregulated expression of FERRITIN and VACUOLAR IRON TRANSPORTER 2 (OsVIT2), whereas NATURAL RESISTANCE-ASSOCIATED MACROPHAGE 1 (OsNRAMP1) expression is upregulated to promote Fe efflux from the vacuoles. Chlorophyll content is increased by upregulating EARLY LESION LEAF 1 (ELL1) and NITRATE REDUCTASE 2 (OsNR2) expression and modulating carbohydrate metabolism to enhance leaf photosynthesis. In the roots, deoxymugineic acid (DMA) synthesis and efflux are promoted by upregulating the expression of NICOTIANAMINE SYNTHASE 1 (OsNAS1), OsNAS2, and TRANSPORTER OF MUGINEIC ACID FAMILY PHYTOSIDEROPHORES 1 (OsTOM1), whereas PLASMA MEMBRANE H⁺-ATPASE 4 (OsA4) expression is upregulated to enhance H⁺ secretion, thereby improving Fe availability in the rhizosphere. Additionally, OsABF1, OsGA2oxs, and OsEUI1 are overexpressed to suppress gibberellin (GA) synthesis and promote GA inactivation, thereby reducing root GA content and mitigating its negative regulatory effects on Fe deficiency responses. Furthermore, Fe deficiency response in the roots is enhanced by modulating the cytokinin (CTK), jasmonic acid (JA), and ethylene (ETH) signaling pathways. Consequently, the tolerance of transgenic SY and SI rice seedlings to Fe deficiency stress is significantly improved