4.1 Response of osmotic adjustments to remedial fertilizer after low temperature stress at jointing stage
Carbohydrates play a crucial role in freezing tolerance (Livingston et al. 2006). Simple sugars, such as trehalose, sucrose, and raffinose, are especially correlated with enhanced cold tolerance (Kaplan et al. 2006; Hassan et al. 2021). In this study, significant increases in soluble sugar concentration in wheat under treatment without fertilizer amendment were observed, especially under the treatment T72N0 (Fig. 2A), indicating that wheat plants adapted to cold stress by increased soluble sugar concentration. Salicylic Acid-treated wheat enhanced total soluble sugar contents under low-temperature conditions (Wang et al. 2020). In our study, the concentration of soluble sugars were reduced from 13.2–20.6%, from 14.7–24%, and from 7–13.2% under T24, T48, and T72 with increased remedial fertilizer rate. These findings imply that remedial fertilizer after low temperature stress at jointing stage is an efficient way to increase sucrose inversion.
Another essential protective substance in the plant response to abiotic stress is proline. The level of proline was correlated with wheat low-temperature tolerance (Dörffling et al.2009). In the present study, proline accumulation was enhanced on the 10th day after low-temperature stress without remedial fertilizer compared with the control and increased dramatically by 88.6% under T24N0, 107% under T48N0, and 130% under T72N0 (Fig. 2C, 2D). Proline may act as an osmolyte and function as a compatible ROS scavenger, protecting the plant from such oxidative stress. Its accumulation might balance the cell redox status and buffer the cytosolic pH (Majláth et al.2012). High concentrations of proline can increase tissue turgor, advance osmotic regulation, and enhance plant resistance to low temperature (Li et al. 2017). In this study, the proline concentration was reduced significantly with increased remedial fertilizer at the same low-temperature duration, which demonstrated that remedial urea might contribute to the balance of proline metabolism and the growth recovery of the wheat plants. In summary, the lower soluble sugar and proline concentrations in wheat were likely to result in osmoregulatory recovery after remedial urea application, which ultimately contributed to faster recovery and growth of wheat plants.
4.2 Hormone changes of remedial fertilizer after low temperature stress at jointing stage
Plant endogenous hormones, such as ABA, GA, and CKS, function as signaling molecules and participate in cold resistance (Sun et al. 2009; Vaseva et al. 2009; Wang et al. 2018). Low temperature-induced ABA accumulation in winter wheat at booting stage altered the activity of enzymes related to sucrose metabolism, which led to sucrose synthesis and accumulation in the young ears, thus causing yield losses (Zhang et al. 2019). In this study, we also noticed that soluble sugar and ABA concentrations were increased in abundance after low-temperature stress from 24 h to 72 h (Fig. 2A, 3A). Lower GA3 content and higher ABA content in the seeds after chilling injury was previously found to inhibit seed germination, while treating the seeds with GA3 could mitigate the low-temperature injury to seed germination (Hou 2003). In this study, the GA3 concentration changed conversely to the concentration of ABA not only after low temperature (Fig. 3C), but also after the remedial urea amendment. In addition, remedial urea significantly maintained lower levels of endogenous ABA and ZR and higher GA3 levels, which enhanced the wheat growth rate. These results are well in accordance with the observations of Yang et al. (2013), who reported that low-temperature conditioning alleviated injury in kiwifruit by promoting higher ABA/GA3 ratios.
In the present study, the levels of ABA and ZR in the treatment without remedial fertilizer gradually increased with the longer low-temperature duration at jointing stage, which are typical responses of wheat to low-temperature stress (Fig. 3A, 3B). These results suggested that wheat changed the balance of these hormones to adapt to low-temperature stress. Remedial urea was conducive to alleviating the low-temperature damage to wheat and improving wheat growth via the reduced ABA as well as ZR and increased GA3 concentrations following low-temperature stress at jointing stage.
4.3 Contribution of remedial fertilizer after freezing injury to plant morphology and grain yield
The warming of global mean winter temperatures promotes wheat growth in the previous winter, enhancing cold vulnerability in the next spring (Li et al. 2016). Low temperature can remarkably reduce the rates of wheat growth and development, resulting in changes in morphological characters, such as leaf area (Valluru et al. 2012), ear number, and grain number (Thakur et al. 2010; Li et al. 2017). Limin and Fowler (2000) reported that the cell size of wheat decreased and the young and new leaves became shorter and narrower following cold acclimation. In the present study, the degree of cold injury to the wheat plants and the freezing injury index were all increased from 0.3 to 0.5 with increased low temperature duration at jointing stage, resulting in lower plant height, especially the first and second upper internode length and ear length. Our results are consistent with the report of Li et al. (2017), who found that chilling stress treatment significantly decreased plant height and leaf area in hexaploid wheat. These results indicated that low temperatures affected wheat development mainly by reducing the growth rate, resulting in reduced plant height.
Several cold hardening experiments suggested that endogenous hormones, such as IAA and CKs, may actively participate in the control of plant growth under low-temperature stress (Majláth et al. 2012). Our findings confirmed that there was a strong negative correlation between plant height as well as the upper two internode length and related physiological parameters (Table 3), including proline, soluble sugar, ABA, and ZR concentrations, while GA3 concentration was positively correlated with these. These data also confirmed that internode elongation was inhibited by higher ABA and ZR levels and lower GA3 level under low temperature. In contrast, the balance among these hormones improved with remedial urea application after low-temperature stress, resulting in greater plant height (Fig. 1). Remedial fertilizer after cold stress at jointing stage mainly promoted the length of the two upper internodes and ear length, ultimately increasing the plant height.
Low temperature not only inhibits wheat growth and development but also results in the loss of grain yield. In Kansas, an additional day of freezing temperature in the spring was associated with a 3.3% yield reduction over the 1985–2013 period (Tack et al. 2015). In certain regions of Australian wheat belt, a high risk of ≥ 10% yield losses result from post-head-emergence frosts (Zheng et al. 2015). The wheat yield was reduced by 42.5–59.8% under low temperature (−2°C/−8°C, day/night) from 24 h to 72 h at tillering stage in Jiangsu province in China (Li et al. 2017). In the present study, the wheat yield was reduced by 49.42–65.11% under low temperature (3°C/−4°C, day/night) from 24 h to 72 h at jointing stage. Internode extension and dry matter accumulation were restricted and the spikelet was killed, and thus the grain yield was also decreased due to low temperature during stem elongation (Whaley et al. 2004). Our findings also demonstrated that the reduced number of fertile tillers and grain number per spike were primarily associated with the loss of yield under cold stress at jointing stage.
The cold resistance of wheat was correlated with the levels of expression of TaEXPB7-B in the tillering nodes, which was up-regulated by both low-temperature and ABA (Feng et al. 2019). As a result, young and new tillers were generated rapidly after cold stress at jointing stage (Liu et al. 2019). We also observed this phenomenon in our study. Treatment with remedial urea after low temperature at jointing stage generated more new tillers and enhanced grain yield. Of course, the yield under treatment with remedial urea could not wholly restore and reach the level in natural conditions. The principal component analysis results indicated that grain yield was positively correlated with GA3. The GA3 concentration was higher 23.68% –52.65%, 16.67–41.4%, 25.6–79.8% with the increased rate of remedial urea than those with T24N0, T48N0 and T72N0, which could benefit from promoting wheat growth, inducing new generated tillers, and internode extension. With increased fertilizer application rate under the same treatment duration, the height of the wheat plants was better restored and the loss of grain yield was lessened.
These findings indicated that remedial fertilizer could increase the growth recovery of the cold-damaged wheat plants and reduce the loss of wheat yield. At jointing stage, considering the recovery effect and nitrogen partial factor productivity, 105 kg ha−1 urea is recommended for nitrogen amendment when wheat plants are damaged slightly and the freezing injury index is about 0.3. When the freezing injury index is about 0.5, 150 kg ha−1 urea is suggested. When the freezing injury index is about 0.7, 225 kg ha−1 urea is recommended for recovering wheat growth after severe cold damage. Our findings can offer useful and practical approaches for alleviating the impacts of low temperatures in spring on wheat production. Of course, we need to think about the comprehensive income, including the remedial urea costs, the price of wheat, the negative effect of remedial urea on weak-gluten wheat quality, because the grain protein content will be improved with the remedial urea application after jointing stage.