The impact of the absence of aliphatic glucosinolates on water transport under salt stress in Arabidopsis thaliana

Abstract

Members of the Brassicaceae are known for their contents of nutrients and health-promoting phytochemicals, including glucosinolates. Exposure to salinity increases the levels of several of these compounds, but their role in abiotic stress response is unclear. The effect of aliphatic glucosinolates on plant water balance and growth under salt stress, involving aquaporins, was investigated by means of Arabidopsis thaliana mutants impaired in aliphatic glucosinolate biosynthesis, which is controlled by two transcription factors: Myb28 and Myb29. The double mutant myb28myb29, completely lacking aliphatic glucosinolates, was compared to wild type Col-0 (WT) and the single mutant myb28. A greater reduction in the hydraulic conductivity of myb28myb29 was observed under salt stress, when compared to the WT and myb28; this correlated with the abundance of both PIP1 and PIP2 aquaporin subfamilies. Also, changes in root architecture in response to salinity were genotype dependent. Treatment with NaCl altered glucosinolates biosynthesis in a similar way in WT and the single mutant and differently in the double mutant. The results indicate that short-chain aliphatic glucosinolates may contribute to water saving under salt stress.

Keywords: Arabidopsis thaliana; Brassicaceae; glucosinolates; hydraulic conductance; plasma membrane intrinsic protein.

Figure 1

Root hydraulic conductance, L₀, (mg h⁻¹ MPa⁻¹ g⁻¹ DW) (A), relative water content (RWC) (B) and whole plant transpiration (mg m² s⁻¹) (C) of Arabidopsis thaliana ecotype Col-0 wild type (WT), single (myb28) and double (myb28myb29) knockout mutants under non-saline (0 mM NaCl) and saline (100 mM NaCl) conditions. Mean values ± standard errors are shown (n = 10). Mean values with different letters represent significant (P < 0.05) differences according to the Tukey test.

Figure 2

Immunodetection of PIP1 (A) and PIP2 (B) homologs in the root plasma membrane (PM) of Arabidopsis thaliana ecotype Col-0 wild type (WT), single (myb28) and double (myb28myb29) knockout mutants under non-saline (0 mM NaCl) and saline (100 mM NaCl) conditions. Total PM was separated by SDS-PAGE and probed with antibodies against AtPIP1 and AtPIP2. The PIP1 and PIP2 protein amounts (monomers and dimers) were quantified using the “Quantity one” program from Bio-Rad Laboratories. Mean values are shown (n = 3). Mean values with different letters represent significant (P < 0.05) differences according to the Tukey test. The scanned bands were normalized by calculating the ratio to the corresponding Coomassie-stained aquaporin band (29 KDa) ensuring that different intensities were not due to different loaded protein amounts.

Figure 3

Identification of intact glucosinolates by HPLC-DAD-ESI-MSn in the leaves of Arabidopsis thaliana ecotype Col-0 wild type (WT) (A), single (myb28) (B) and double (myb28myb29) (C) knockout mutants under non-saline (0 mM NaCl) conditions.

Figure 4

Total aliphatic (A), total indolic (B) and total glucosinolates (C) (mg g⁻¹ DW) in the leaves of Arabidopsis thaliana ecotype Col-0 wild type (WT), single (myb28) and double (myb28myb29) knockout mutants under non-saline (0 mM NaCl) and saline (100 mM NaCl) conditions. Mean values ± standard errors are shown (n = 20). Mean values with different letters represent significant (P < 0.05) differences according to the Tukey test.

Figure 5

Total phenolic compounds (mg g⁻¹ DW), as the sum of chlorogenic acid, flavonoids and sinapic acid derivates, in the leaves of Arabidopsis thaliana ecotype Col-0 wild type (WT), single (myb28) and double (myb28myb29) knockout mutants under non-saline (0 mM NaCl) and saline (100 mM NaCl) conditions. Mean values ± standard errors are shown (n = 20). Mean values with different letters represent significant (P < 0.05) differences according to the Tukey test.