# Different application methods of humic acid and zinc differentially regulate osmotic balance and C-repeat binding factor pathways in cold-stressed oat (Avena sativa L.)

## TL;DR

This study shows that applying humic acid and zinc to oat plants helps them better handle cold stress by improving water balance and activating cold defense genes.

## Contribution

The study reveals that specific application methods of humic acid and zinc enhance cold tolerance in oat plants through osmotic and gene regulatory pathways.

## Key findings

- Soil application of HA+Zn and combined seed-soil treatments improved membrane durability and water content in cold-stressed oat plants.
- CBF gene expression was most strongly upregulated under soil-applied HA+Zn, indicating enhanced cold defense activation.
- Cold-tolerant and cold-sensitive oat cultivars responded differently to HA and Zn treatments under low temperatures.

## Abstract

The present study investigated the effects of different humic acid (HA) and zinc (Zn) application methods on membrane durability index (MDI), leaf relative water content (RWC), turgor loss (TL), proline content (PC), and C-repeat binding factor (CBF) gene expressions in oat plants exposed to various low temperatures. For this purpose, two oat cultivars—Albatros (cold-sensitive) and Checota (cold tolerant)—were grown under controlled conditions with HA and Zn applied to the seeds or soil, either individually or in combination, until the 3–4 leaf stage. The plants were subsequently exposed to temperatures of 4 °C, 0 °C, −5 °C, −10 °C, and −15 °C for 24 h each. The results indicated that the application methods of HA and Zn substantially influenced the plants’ responses to low temperature. Among the treatments, soil application of HA+Zn (SA_HA+Zn), seed priming with HA combined with soil-applied Zn (SP_HA+SA_Zn), and seed priming with Zn combined with soil-applied HA (SP_Zn+SA_HA) provided the greatest protection against cold stress, as evidenced by improved MDI, RWC, TL, and PC levels. Gene expression analyses further revealed that low temperatures upregulated the CBF genes and the related regulatory genes VRN1 and ZAT12, with the strongest induction observed under SA_HA+Zn, suggesting that this combined approach more effectively activates the plant’s cold defense mechanisms.