Nitrate-dependent changes in the primary and lateral root growth in wheat seedlings require the coordinated action of auxin, calcium and nitric oxide.

Nitrate (NO), besides serving as a major N source, also acts as a signalling molecule in plant growth and development. Studies on NO dependent regulation of root growth in wheat (Triticum aestivum) are mostly limited to morphophysiological changes, while the underlying signalling mechanisms remain largely unexplored. To bridge this gap, the present study aims to get a mechanistic understanding of the NO dependent regulation of root growth in wheat seedlings. For this, uniformly germinated two days old wheat seedlings were exposed to nitric oxide (NO) donor sodium nitroprusside (SNP), auxin source Indole-3-butyric acid (IBA), calcium chloride as source of calcium (Ca), NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), polar auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) and calcium chelator ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) with NO as a major determinant. After seven days, root traits were analyzed, and a tissue localization assay was performed using Fluo-3 AM, DAF-FM, and salkowski reagents to visualize the distribution of Ca, NO, and indole-3-acetic acid (IAA) in root tissues, respectively. The study reveals that the cross-talk of nitric oxide (NO), auxin and calcium (Ca) modulates NO regulated root growth in wheat seedlings. The changes in cytosolic Ca concentration ([Ca]) are a major mediator in the regulation of root growth. High NO (5 mM) in combination with exogenous IBA and Ca suppresses the root growth, with ethylene acting downstream of [Ca]. We observed a synergistic effect between NO and endogenous IAA (Indole-3-acetic acid) in lateral root (LR) growth. In LRs, the external NO enhances the NO production, which is further augmented by the elevated [Ca] levels. Our results indicate that endogenous IAA plays a pivotal role in regulating root hair development on LR with NO and [Ca] functioning downstream of the signalling cascade. However, the high NO was found to counteract the root hair formation by importing the shoot-derived auxin. These findings provide valuable insights into the intricate signalling interactions between nitric oxide, auxin, and calcium in NO regulated root development in wheat, with potential targets for enhancing nutrient uptake efficiency. Further work is necessary to identify downstream signalling components and examine how shoot-to-root signalling modulates the root architectures under different NO regimes.