Contrasting roles of GmNAC065 and GmNAC085 in natural senescence, plant development, multiple stresses and cell death responses

Abstract

NACs are plant-specific transcription factors involved in controlling plant development, stress responses, and senescence. As senescence-associated genes (SAGs), NACs integrate age- and stress-dependent pathways that converge to programmed cell death (PCD). In Arabidopsis, NAC-SAGs belong to well-characterized regulatory networks, poorly understood in soybean. Here, we interrogated the soybean genome and provided a comprehensive analysis of senescence-associated Glycine max (Gm) NACs. To functionally examine GmNAC-SAGs, we selected GmNAC065, a putative ortholog of Arabidopsis ANAC083/VNI2 SAG, and the cell death-promoting GmNAC085, an ANAC072 SAG putative ortholog, for analyses. Expression analysis of GmNAC065 and GmNAC085 in soybean demonstrated (i) these cell death-promoting GmNACs display contrasting expression changes during age- and stress-induced senescence; (ii) they are co-expressed with functionally different gene sets involved in stress and PCD, and (iii) are differentially induced by PCD inducers. Furthermore, we demonstrated GmNAC065 expression delays senescence in Arabidopsis, a phenotype associated with enhanced oxidative performance under multiple stresses, higher chlorophyll, carotenoid and sugar contents, and lower stress-induced PCD compared to wild-type. In contrast, GmNAC085 accelerated stress-induced senescence, causing enhanced chlorophyll loss, ROS accumulation and cell death, decreased antioxidative system expression and activity. Accordingly, GmNAC065 and GmNAC085 targeted functionally contrasting sets of downstream AtSAGs, further indicating that GmNAC85 and GmNAC065 regulators function inversely in developmental and environmental PCD.

Figure 1

Phylogenetic reconstruction of Arabidopsis thaliana and soybean (Glycine max) NAC-SAGs. The deduced amino acid sequences of previously described NAC-SAGs of Arabidopsis were used as prototypes to retrieve their putative orthologous genes (GmNAC-SAGs) from the soybean genome. The phylogenetic reconstruction resulted in seven distinct groups with stable collapsed branches. Phylogenetic relationships were established by the neighbor-joining statistical method with 10,000 bootstraps, and the tree was rendered using the MABL interface.

Figure 2

Expression pattern of GmNAC-SAGs under different stresses and bleomycin-induced cell death in soybean. (A) Heatmap of GmNAC-SAGs expression in soybean exposed to different stresses. Transcriptome-wide data from publically accessed RNAseq repository on NCBI were accessed, and the expression of GmNAC-SAGs investigated under drought (moderate and severe), oxidative stress, biotic stresses (fungi and insect attack), and senescence (ethylene-induced and age-induced) conditions. Fold variation values from DEGs were recovered and converted into the heatmap. (B) Expression profile of GmNAC-SAGs in soybean seedlings exposed to bleomycin-treatment (420 mM) for 24 h.

Figure 3

Tissue-specific expression of GmNAC065 and GmNAC085 in Williams 82 soybean seedlings under normal developmental conditions and different stresses. (A) Expression pattern of GmNAC065 and GmNAC085 in soybean vegetative and reproductive stages. Gene expression was determined by RT-qPCR (data available in Supplementary Figure 2A) and calculated using 2^−ΔCt method and ELF1A as the endogenous normalizer gene. (B) Expression pattern of GmNAC065 and GmNAC085 in soybean seedlings subjected to simulated drought (PEG 8000—10% w/v; air-dry and ABA—150 mM), ER stress (Tun—5 µg/mL), and biotic stress (SA—75 µM). The gene expression level was analyzed after 2 h, 4 h and 12 h treatments in leaves and roots by RT-qPCR (data available in Supplementary Figure 2B). Fold variation was calculated using the 2^−ΔΔCt method in three biological replicates and two technical replicates and normalized by Z-score considering gene expression in untreated plants.

Figure 4

Phenotypical characterization of three homozygous independent lines of Arabidopsis overexpressing GmNAC065 and GmNAC085. (A) Phenotypical characterization of GmNAC065-OX and GmNAC085-OX lines (L1, L2 and L3) during the vegetative stage and the onset of the reproductive stage. Plants were analyzed up to 42 days after germination (DAG) in the vegetative stage and up to 56 DAG in the reproductive stage. 49 DAG was considered the onset of the reproductive stage, hallmarked by the inflorescence’s emergence. (B) Phenotypical characterization of GmNAC065-OX and GmNAC085-OX lines in the reproductive stage. (C) Phenotypical characterization of transgenic Arabidopsis lines during senescence. All experiments were performed with plants under normal development, cultivated in a growth chamber with standard settings. Scale bars = 0.5 cm.

Figure 5

Expression of GmNAC065 and GmNAC085 leads to H₂O₂ accumulation and lipid peroxidation. (A) DAB-leaf staining to detect H₂O₂ accumulation in Arabidopsis leaves submitted to different stresses (PEG 8000—10% w/v; Tun—5 µg/mL; SA—75 µM) for 24 h. (B) TBA-reactive compounds quantification in GmNAC065-OX and GmNAC085-OX lines under different stresses. Solid bars show data from untreated plants. Upper bars indicate standard error (95% of confidence). Uppercase letters indicate significant differences among control samples and lowercase letters indicate significant differences among treated samples by the Tukey's test (p < 0.05, n = 3).

Figure 6

Expression profiles and activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX) in GmNAC065-OX and GmNAC085-OX lines under different stress conditions. (A) Expression profile of CAT, SOD, and APX in transgenic Arabidopsis lines after 24 h of stresses. Fold variation data were normalized according to the expression levels of the control samples (FV = 1.0). (B) Enzymatic activity of CAT, SOD, and APX in GmNAC065-OX and GmNAC085-OX plants under different stresses. Drought, ER stress, and biotic stress were simulated by PEG 8000—10% w/v; Tun—5 µg/mL; and SA—75 µM treatments, respectively. Solid bars show data from untreated plants. Upper bars indicate standard error (95% of confidence). Uppercase letters indicate significant differences among control samples, and lowercase letters indicate significant differences among treated samples by the Tukey's test (p < 0.05, n = 3).

Figure 7

Metabolite content in transgenic Arabidopsis lines overexpressing GmNAC065 and GmNAC085 under different stress conditions. (A) Chlorophyll content. The chlorophyll content was accessed spectrophotometrically from ethanolic plant extracts. (B) Protein decay ratio. After stress, the protein decay ratio was calculated according to the total protein content, determined by the Bradford method. The protein content of untreated plants was considered 100% and the degradation ratio was expressed as the percentage of protein relative to the control. (C) Carotenoid content. The carotenoid content was determined spectrophotometrically from ethanolic plant extracts. (D) Anthocyanin content. Anthocyanin content was determined spectrophotometrically from ethanolic plant extracts. (E) Soluble sugar content was determined by the DNS-reducing method. Samples were subjected to the DNS reaction, and sugar concentration was determined based on a glucose standard curve. Drought, ER stress, and biotic stress were simulated by PEG 8000—10% w/v; Tun—5 µg/mL; and SA—75 µM treatments for 24 h, respectively. Solid bars show data from untreated plants. Upper bars indicate standard error (95% of confidence). Uppercase letters indicate significant differences among control samples, and lowercase letters indicate significant differences among the treated samples by the Tukey's test (p < 0.05, n = 3).

Figure 8

Cell death extent in leaves and roots of GmNAC065-OX and GmNAC085-OX plants. (A) Evans blue staining of GmNAC065-OX and GmNAC085-OX leaves under different stresses. 24 h-stressed and non-stressed leaves were exposed to the Evans dye staining solution for 8 h. The intense blue color indicates the extent of cell death. (B) Propidium iodide (PI) root-staining in transgenic plants ectopically expressing GmNAC065 and GmNAC085. Plants were stressed with PEG 8000—10% w/v; Tun—5 µg/mL; and SA—75 µM for 24 h and the roots were subjected to PI-staining solution for 24 h until confocal microscopy scanning. PI stains the cell wall of living cells whereas dead cells show nuclei and cytoplasm stain. Yellow arrows indicate regions with extensive cell death. Scale bars = 20 µm.

Figure 9

AtSAGs expression in GmNAC065-OX and GmNAC085-OX plants under different stresses. (A) Expression levels of ANAC083/VNI2 and ANAC072, a negative and positive regulator of senescence in Arabidopsis, and the putative orthologous of GmNAC065 and GmNAC085, respectively. (B) Expression levels of stress marker genes and AtSAGs in transgenic lines ectopically expressing GmNAC065 and GmNAC085. ACT2 was used as the endogenous control gene. Relative gene expression was quantified using the comparative 2^−ΔΔCt method. Upper bars indicate standard error (95% of confidence), and the different letters indicate significant differences among the lines by the Tukey's test (p < 0.05, n = 3).

Figure 10

Schematic representation of the gene regulatory network coordinated by environmental stimuli, plant hormones, and AtNAC-SAGs. The different environmental stimuli culminate in plant hormone signaling, which activates several SAGs in response to multiple stresses. Consequently, the expression of these SAGs triggers an age- or -environmental cell death. GmNAC085 up-regulates ATAF1, ORE1, and AtNAP, involved in integrating ABA, age, and ROS signals. Increased expression of these genes results in the activation of several downstream SAGs involved in the degradation of chlorophyll and other pigments, which confer accentuated senescence phenotype. Contrastingly, the expression of these genes is attenuated by GmNAC065 leading to a better plant performance under multiple stresses and a delayed senescence phenotype. Adjusted from Bengoa-Luoni et al..