A CAM-Related NF-YB Transcription Factor Enhances Multiple Abiotic Stress Tolerance in Arabidopsis

Abstract

Abiotic stresses often occur simultaneously, and the tolerance mechanisms of plants to combined multiple abiotic stresses remain poorly studied. Extremophytes, adapted to abiotic stressors, might possess stress-adaptive or -responsive regulators that could enhance multiple abiotic stress resistance in crop plants. We identified an NF-YB transcription factor (TF) from the heat-tolerant obligate Crassulacean acid metabolism (CAM) plant, Kalanchoe fedtschenkoi, as a potential regulator of multiple abiotic stresses. The KfNF-YB3 gene was overexpressed in Arabidopsis to determine its role in multiple abiotic stress responses. Transgenic lines exhibited accelerated flowering time, increased biomass, larger rosette size, higher seed yield, and more leaves. Transgenic lines had higher germination rates under combined NaCl, osmotic, and water-deficit stress treatments compared to control plants. They also showed enhanced root growth and survival under simultaneous NaCl, osmotic, water-deficit, and heat stress conditions in vitro. Interestingly, potted transgenic lines had higher survival rates, yield, and biomass under simultaneous heat, water-deficit, and light stresses compared to control plants. Altogether, these results provide initial insights into the functions of a CAM-related TF and its potential roles in regulating multiple abiotic stress responses. The CAM abiotic stress-responsive TF-based approach appears to be an ideal strategy to enhance multi-stress resilience in crop plants.

Keywords: Crassulacean acid metabolism; abiotic stress; climate change; extremophytes; nuclear factor Y; transcription factor.

Figure 1

Phylogenetic analysis of the KfNF-YB3 gene. (A) Multiple sequence alignment showing the conserved NF-YB binding domain among KfNF-YB3 and several abiotic-responsive NF-YB transcription factors from different plant species (* = highly conserved, : = moderately conserved, . = less conserved). (B) Secondary protein structures of KfNF-YB3 compared with NF-YBs from other plant species, with secondary structures presented above the alignment (helix motifs indicated by α1–αC and loops by L1–L3). (C) Phylogenetic tree illustrating the evolutionary relationship of KfNF-YB3 with other abiotic stress-responsive genes in the NF-Y family.

Figure 1

Phylogenetic analysis of the KfNF-YB3 gene. (A) Multiple sequence alignment showing the conserved NF-YB binding domain among KfNF-YB3 and several abiotic-responsive NF-YB transcription factors from different plant species (* = highly conserved, : = moderately conserved, . = less conserved). (B) Secondary protein structures of KfNF-YB3 compared with NF-YBs from other plant species, with secondary structures presented above the alignment (helix motifs indicated by α1–αC and loops by L1–L3). (C) Phylogenetic tree illustrating the evolutionary relationship of KfNF-YB3 with other abiotic stress-responsive genes in the NF-Y family.

Figure 2

Phenotype characterization of OxKfNF-YB3 T₃ transgenic lines and Col-0 WT control plants. (A) Representative image of rosette morphology of two-week-old seedlings (n = 30). (B) Rosette size in two-week-old seedlings. (C) Representative image of rosette morphology of five-week-old seedlings. (D) Rosette size of five-week-old seedlings. Col-0 WT = gray, OxKfNF-Y-1 = red, OxKfNF-Y-2 = blue, OxKfNF-Y-16 = green, OxKfNF-Y-17 = orange. n = 30, experiments replicated three times, values represent means ± SE, ** adjusted p < 0.01.

Figure 3

Growth characteristics of five-week-old T₃ transgenic seedling lines and Col-0 WT control seedlings. (A) Plant height. (B) Number of leaves. (C) Shoot biomass. (D) Seed yield. (E) Flowering time. Col-0 WT = gray, OxKfNF-Y-1 = red, OxKfNF-Y-2 = blue, OxKfNF-Y-16 = green, OxKfNF-Y-17 = orange. n = 30, experiments replicated three times, values represent means ± SE, ** adjusted p < 0.01.

Figure 4

Water-use efficiency (WUE) of four-week-old OxKfNF-YB3 T₃ transgenic lines and Col-0 WT control plants. (A) Representative image of single seedlings of transgenic lines and Col-0 WT plants after four weeks of WUE assay (n = 30). (B) Quantification of integrated WUE after four weeks. Col-0 WT = gray, OxKfNF-Y-1 = red, OxKfNF-Y-2 = blue, OxKfNF-Y-16 = green, OxKfNF-Y-17 = orange. n = 30, values represent means ± SE, ** adjusted p < 0.01.

Figure 5

Water-deficit stress treatment of OxKfNF-YB3 T₃ transgenic lines and Col-0 WT control plants. (A,B) Representative image of overexpression lines and Col-0 WT control seedlings recorded at different stages during a 15-day water-deficit stress treatment (n = 30). (C) Survival rate after rewatering. (D) Shoot biomass after rewatering. (E) Seed yield after rewatering. Col-0 WT = gray, OxKfNF-Y-1 = red, OxKfNF-Y-2 = blue, OxKfNF-Y-16 = green, OxKfNF-Y-17 = orange. n = 10, experiments replicated three times, values represent means ± SE, ** adjusted p < 0.01.

Figure 6

Hypocotyl length elongation in OxKfNF-YB3 transgenic lines and Col-0 WT control plants under heat stress treatments. (A) Representative set up of in vitro assays of NaCl + PEG and mannitol + PEG, combined with heat stress. (B) No stress treatment. (C) Heat stress. (D) NaCl + PEG + heat stress. (E) Mannitol + PEG + heat stress. Col-0 WT = gray, OxKfNF-Y-1 = red, OxKfNF-Y-2 = blue, OxKfNF-Y-16 = green, OxKfNF-Y-17 = orange. n = 30, experiments replicated three times, values represent means ± SE, ** adjusted p < 0.01.

Figure 7

Seed germination of OxKfNF-YB3 transgenic lines and Col-0 WT control plants under individual and combined stress treatments. (A) Representative set up of in vitro assays of 250 mM mannitol, 200 mM NaCl, −0.5 MPa PEG-8000, NaCl + PEG, mannitol + PEG, and NaCl + mannitol + PEG for 16 days. (B) NaCl stress. (C) PEG stress. (D) Mannitol stress (250 mM). (E) NaCl + PEG stress. (F) Mannitol + PEG stress. (G) NaCl + mannitol + PEG stress. (H) Combined stress assay post-germination (16 d) in MS media with 200 mM NaCl and PEG-8000. (I) Root length. (J) Biomass. Col-0 Col-0 WT = gray, OxKfNF-Y-1 = red, OxKfNF-Y-2 = blue, OxKfNF-Y-16 = green, OxKfNF-Y-17 = orange. n = 30, experiments replicated three times, values represent means ± SE, ** adjusted p < 0.01.

Figure 7

Seed germination of OxKfNF-YB3 transgenic lines and Col-0 WT control plants under individual and combined stress treatments. (A) Representative set up of in vitro assays of 250 mM mannitol, 200 mM NaCl, −0.5 MPa PEG-8000, NaCl + PEG, mannitol + PEG, and NaCl + mannitol + PEG for 16 days. (B) NaCl stress. (C) PEG stress. (D) Mannitol stress (250 mM). (E) NaCl + PEG stress. (F) Mannitol + PEG stress. (G) NaCl + mannitol + PEG stress. (H) Combined stress assay post-germination (16 d) in MS media with 200 mM NaCl and PEG-8000. (I) Root length. (J) Biomass. Col-0 Col-0 WT = gray, OxKfNF-Y-1 = red, OxKfNF-Y-2 = blue, OxKfNF-Y-16 = green, OxKfNF-Y-17 = orange. n = 30, experiments replicated three times, values represent means ± SE, ** adjusted p < 0.01.

Figure 8

Survival rate of OxKfNF-YB3 transgenic seedlings and Col-0 WT control seedlings under combined multiple stress treatments. (A) Representative set up of in vitro assays with 250 mM mannitol, 200 mM NaCl, −0.5 MPa PEG-8000, NaCl + PEG, mannitol + PEG, and NaCl + mannitol + PEG combined with heat stress. (B) Heat stress. (C) NaCl + heat stress. (D) Mannitol + heat stress. (E) PEG + heat stress. (F) NaCl + PEG + heat stress. (G) NaCl + mannitol + heat stress. Col-0 WT = gray, OxKfNF-Y-1 = red, OxKfNF-Y-2 = blue, OxKfNF-Y-16 = green, OxKfNF-Y-17 = orange. n = 30, experiment replicated three times, values represent means ± SE, ** adjusted p < 0.01.

Figure 9

Combined multiple stress treatment with water deficit, heat, and light in OxKfNF-YB3 T₃ transgenic lines and Col-0 WT control plants. (A,B) Representative images of overexpression lines and Col-0 WT control plants recorded at different stages during the combined multiple stress treatment (n = 30). (C) Survival rate after rewatering. (D) Shoot biomass after rewatering. (E) Seed yield after rewatering. Col-0 WT = gray, OxKfNF-Y-1 = red, OxKfNF-Y-2 = blue, OxKfNF-Y-16 = green, OxKfNF-Y-17 = orange. n = 30, experiment conducted once, values represent means ± SE, ** adjusted p < 0.01.