Osmoprotectant accumulation patterns in mustard (Brassica juncea) under progressive water stress

Introduction

Nearly 68% of the world's mustard crop grows under rainfed conditions where drought can reduce seed yields by 30-55% [1] . Brassica juncea ranks among the most important global oilseed crops, yet the biochemical mechanisms behind cultivar-level water deficit tolerance remain incompletely mapped [2] . Plants respond to water stress through osmotic adjustment active accumulation of compatible solutes that lower cellular osmotic potential without disrupting enzymes [3] . Key osmoprotectants include proline (an osmolyte and ROS scavenger) [4] , glycine betaine (stabilizes proteins and membranes) [5] , trehalose (protects membranes) [6] , and soluble sugars. Most previous work examined one or two osmolytes in isolation [7,8] , missing combined contributions and potential synergies. This research aimed to: (i) track accumulation kinetics of five osmoprotectant classes across three stress levels in four B. juncea cultivars; (ii) determine each osmolyte's relative contribution to total osmotic adjustment; and (iii) identify which accumulation pattern best predicts seed yield retention.

Research area and environmental conditions

The experiment was conducted in the controlled-environment glasshouses at ETH Zurich, Zurich, Switzerland (47°22'N, 8°33'E; 408 m elevation) between October 2022 and March 2023, with a repeat season from October 2023 to March 2024. Glasshouse temperature was maintained at 22/16 °C (day/night) with natural light supplemented by sodium lamps to provide a 14-hour photoperiod. Relative humidity averaged 55±8% [9] .

Materials and Methods

Materials

Seeds of four B. juncea cultivars were sourced from the Swiss Federal Seed Collection. Analytical standards (≥97%) were from Sigma-Aldrich; ninhydrin and Grieve's reagents from Merck. Research was approved by ETH Plant Sciences Ethics Review (Protocol ETH-PS/2022-017, August 2022).

Methods

Proline was estimated by acid-ninhydrin [11] ; glycine betaine by periodide colorimetry [12] ; trehalose enzymatically after 80% ethanol extraction; soluble sugars by anthrone method; free amino acids by ninhydrin-cadmium acetate. Total osmotic adjustment was the difference in osmotic potential at full turgor between stressed and control plants (Wescor 5520 osmometer). ANOVA was run in R 4.3 with Tukey's HSD at p ≤ 0.05.

Field experimental design

A factorial randomized complete block design was used with four cultivars (Pusa Bold, Varuna, RH-749, PM-25) and three moisture regimes (100%, 50%, and 25% FC) in four replications. Plants were grown in 12-L pots with a sandy loam substrate. Water stress was imposed at flowering onset (50 DAS) by adjusting irrigation to target FC levels, monitored with TDR soil moisture sensors. Leaf and stem tissue samples were collected at 10-day intervals from 50 to 90 DAS for osmolyte analysis [10] . Figure 1 shows osmolyte contributions: proline dominated (38.2%), followed by glycine betaine (24.7%) and soluble sugars (16.8%). Figure 2 highlights Varuna's consistently higher accumulation across all osmolytes.

Results

Discussion

Proline's dominance in the osmolyte pool (38.2%) aligns with reports from Brassica napus [3,4] . But proline alone didn't predict tolerance RH-749 accumulated moderate proline yet showed the weakest adjustment. The key was glycine betaine coaccumulation, 1.8-fold higher in tolerant Varuna than in RH-749, reflecting complementary roles: proline scavenges ROS while glycine betaine stabilizes membranes [5,6] . The strong correlation (r = 0.93) between total osmotic adjustment and yield confirms multi-osmolyte accumulation as a better selection criterion than any single metabolite. Trehalose contributed only 11.3% of adjustment but may have disproportionate membrane-protective effects not captured by osmometric measurements [6] .

Limitations

Glasshouse conditions may not reflect field drought where soil heterogeneity and variable evaporative demand create uneven stress. Four cultivars provide a limited genetic base for generalizing osmolyte-tolerance relationships. Root-zone osmolyte dynamics were not measured and could differ from shoot patterns.

Conclusion

Progressive water stress triggered distinct osmoprotectant patterns in B. juncea, with proline and glycine betaine together accounting for over 62% of osmotic adjustment. Varuna achieved the highest adjustment (0.89 MPa) and retained 71.3% of control yield at 25% FC. Proline-glycine betaine coaccumulation emerged as the best tolerance predictor, and breeding programs should select for combined accumulation capacity rather than single osmolytes.

Acknowledgement

AcknowledgementsThis research was supported by institutional funds from ETH Zurich, Department of Environmental Systems Science. The authors thank ETH Zurich for glasshouse access and technical support, and acknowledge laboratory technicians for assistance with osmolyte extraction procedures.

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