For the visual observations, nine branches per each scion/rootstock combination (PP, MC, and KG) for each temperature treatment/duration combination (i.e., -2, 0, 2, and 4°C for 4 and 8 hours) at three stages (stage I, stage II, and stage III) were used. To visually observe and compare the damage after the temperature treatments, branches were divided into two categories: number of branches with live male catkins/terminal bud/green healthy leaves ( Figures 1A–E ) and number of branches dead ( Figures 1F, G ).

Branches of three pecan scion/rootstock combinations (PP, MC, and KG) kept in the growth chamber after different low-temperature treatment (from 8 hours duration) were used for soluble sugar and starch analysis of bark (containing phloem vascular tissue) and wood (containing xylem vascular tissue) tissue. The nine branches per scion-rootstock combinations for each temperature treatment were used. The top section (4–5cm in length from the terminal bud) from each of the branches was used. The wood and bark of each section were separated manually, chopped using pruning shears, and dried in the oven (Isotemp oven Model 655F, Fisher Scientific, USA) for 2–3 days at 75°C. After drying, the samples were kept at room temperature until further processing. The fine powder (25–27 mg) bark and wood tissues were further processed for soluble sugars and starch analysis using colorimetric Anthrone regent method (Kaur et al., 2023).

For soluble sugars and starch, total carbohydrate content was determined based on glucose equivalents and were expressed as mg g^-1 DW (dry weight). The data were analyzed using PROC GLM in Statistical Analysis System (Version 9.4; SAS Institute Inc., Cary, NC). Three-way analysis of variance (ANOVA) was performed to determine the effects of low temperature on soluble sugar and starch concentration in different scion/rootstock combinations at three stages, and the treatment differences were analyzed using the LSMEANS with LINES statement at α = 0.05.

Branches collected from three pecan scion/rootstock combinations at stage I (outer bud scale shed stage) and stage II (a week after outer bud scale shed stage) were observed three weeks after the low temperature treatments ( Figures 2 – 5 ). Post cold treatment most Maramec/Colby (MC) branches produced large leaves and catkins ( Figures 2 , 3B-E ). The Pawnee/Peruque (PP), branches also produced leaves and catkins, but most branches were dead on the day of observations ( Figures 2 , 3G-J ). Kanza/Giles (KG) at all the temperature treatments and controls, had bud break and catkins but catkins were very small ( Figures 3L-N ). KG branches treated for 4 hours at temperatures (4, 2, and 0°C) had better leaf or catkin growth than branches treated for 8 hours. Overall, when branches were treated at stage I, most KG branches had only terminal live buds or small catkins, MC branches had large green leaves, and PP had dead branches.

In KG, most branches were dead after all low-temperature treatments (except 4°C for 4 hours) at stage II ( Figure 4 ). Only a few branches of KG produced leaves. Most MC branches produced leaves after treatment. In PP, most branches treated at -2°C for 4 and 8 hours were dead. The percentage of dead branches declined with higher temperature treatments. In 0°C and 2°C treated, PP branches treated for 4 hours had most of branches produced male catkins and green leaves compared to 8 hours treatment. PP branches treated with 4°C for 4 and 8 hours had more leaves and catkins and less percentage of dead branches. Overall, most KG branches treated at stage II were dead or with terminal green buds only whereas MC had green healthy leaves. In PP, many branches were dead when treated at -2°C, and the percentage of dead branches declined with higher temperature treatments. In PP, most of branches treated at 4°C had live male catkins and leaves.

The branches treated with low temperatures at stage III (early bloom stage) were visually observed after two weeks of treatment ( Figure 5 ). In KG and PP, most of branches had dead leaves and catkins after low-temperature treatments. Whereas most of the MC branches had green leaves or terminal green buds after temperature treatments, but they easily dislodged and fell when touched.

Overall, the branches collected at stage III showed the most damage, followed by stage II, while the branches treated at stage I had the least damage.

The three-way scion/rootstock × treatments × stage interaction was significant in bark soluble sugar, bark starch, woody tissue soluble sugar, and woody tissue starch content ( Table 1 ).

Low spring temperatures damage the soft fresh tissues of developing buds, new leaves, and reproductive organs (Hosseinpour et al., 2018). This study demonstrated branches at stage III early bloom, were the most severely damaged by all low temperatures compared to stage II and stage I samples. Freeze/frost injury is highly dependent on the phenological growth stage of the bud/flower (Aygun and San, 2005; Hosseinpour et al., 2018). In pecans, freeze induced minimal harm to dormant or enlarged buds, but caused significant damage to buds during the leaf expansion (Grauke and Pratt, 1992; Sparks, 1992; Farokhzad et al., 2018). Stage III had expanding leaves and flowers, which made them more susceptible to low temperatures compared to stage I and stage II.

Differences in damage/injury were observed among the scion/rootstock combinations after low temperature exposure. Overall, MC branches had the lowest mortality rates across all three growth stages compared to the other two scion/rootstock combinations. It might be because Maramec was the variety originally selected from the experimental orchards at OSU Cimarron Valley Research Station in Perkins, OK, while the other two varieties, Pawnee and Kanza, were released in the southern state of Texas (Smith, 2002; McCraw et al., 2014). Although all three cultivars are cold hardy and adaptable to the northern area, Maramec exhibits better tolerance to spring freezes. Low temperature tolerance varies among buds/flowers of different cultivars and species (Hosseinpour et al., 2018). For example, the severity of springtime low temperature damage varies among genotypes/cultivars in almonds, apples, and sour cherry (Aygun and San, 2005; Szpadzik et al., 2009; Imani et al., 2012).

There was also a difference in damage between the 4-hour and 8-hour duration temperature treatments. The longer 8-hour low temperature treatments caused more mortality than the 4-hour treatments. The extent of damage has been reported to be was directly related to intensity and duration low temperatures (Larsen, 2010).

The selection of superior cultivars is generally based on climatic adaptation, high production, and good kernel and nut quality. Spring freeze damage is an important limiting factor in tree nut production of pecans, walnut (Khadivi et al., 2019), pistachio (Nobari et al., 2012), almond (Imani et al., 2012). Evaluation and understanding of spring freeze damage among cultivars and rootstocks is important for the selection of trees with a broader climatic adaptation (Grauke and Pratt, 1992; Imani and Mahamadkhani, 2011; Khadivi et al., 2019).

The soluble sugar contents in the three pecan scion/rootstock combinations were different after low-temperature treatments. However, the pattern of change was similar among the combinations. In this study, we observed significant decreases in the bark soluble sugar levels in stages I and stage II of branches treated with low temperatures ( Figure 6 ). The MC bark had significantly reduced soluble sugars after a 4°C temperature treatment at stage I and stage II. It also had better growth than the other two scion/rootstock combinations (PP and KG) after a 4°C treatment. This indicates that MC was less affected by 4°C treatment, and that it utilized its bark and woody tissue soluble sugars for buds, new leave, catkin growth, and development. Similar results were reported in peach after controlled freezing treatments; sugar content was lower after freezing temperature treatments (Yun et al., 2014). However, in our samples from an earlier natural freeze experiment, soluble sugar content was increased after low-temperature conditions (Kaur et al., 2023). This could be explained by the fact that excised branches in growth chambers are no longer attached to trees, limiting sugar translocation from lower plant parts to the more resource demanding shoot apex. In field conditions, higher bark soluble sugar content in apical portions after a freeze could be due to mobilization of sugar from lower plant parts to apical portions to support the higher sugar demand during low temperatures. This is consistent with the suggestion that carbohydrates can enhance cold hardiness or to support metabolic processes that confer cold tolerance (Morin et al., 2007). This is also consistent with the observation that apical bud growth is supported by the carbohydrate content near the shoot apex as well as non-structural carbohydrates transported by the vascular system (Tixier et al., 2017). Our research on pecan secondary buds, which can develop into pistillate and staminate flowers if the primary compound buds are killed, suggests that successful bud burst is promoted by carbohydrate levels stored in both the bark and wood (Zhang et al., 2022, Zhang et al, 2023).

Overall, KG, the most damaged combination compared to MC and PP, had higher soluble sugar content in both the bark and woody tissue. This could be due to differences in the growth stages of different scion/rootstock combinations. We collected branches from KG, MC, and PP on same day; during sampling time KG was a little behind in bud break, compared to more advanced growth on MC and PP, indicating these scion/rootstock combinations were using their sugars rapidly for new growth. Similarly, in controlled chamber conditions after low-temperature treatment, KG had the least growth in terms of bud break and growth, leaves, and catkin formation compared to other scion/rootstock combinations. Overall MC had lower soluble sugar content and had a better bud/leaf/catkin growth at all three growth stages. Similarly, when walnut was subjected to freezing conditions, a significant increase in sugar was observed in tolerant versus sensitive genotypes was observed (Panahi et al., 2021).

In stage I, the starch content in bark samples treated at -2°C, 0°C, 2°C, and 4°C for 8 hours was significantly higher than that of controls except 2°C in KG and PP ( Figure 7 ). As the major carbohydrate reserve in trees starch may be utilized as a secondary source when demand exceeds the current photo-assimilate supply (Bustan et al., 2011). At stage II, in controls KG had significantly higher bark starch content than PP. At stage III in KG, PP, and MC, the starch content in bark samples treated at -2°C was significantly lower than controls, 0°C, and 4°C. At stage III, there were no significant variations among the scion/rootstock combinations in bark starch levels after all temperature treatment and controls. This could be a function of damage and slowed growth after being exposed to low temperatures at stage II and stage III compared to stage I. At the bud break stage, carbohydrates taken up by buds are derived from the mobilization of storage reserves (Alves et al., 2007; Simões et al., 2014). At this stage, glucose is generated from starch degradation or by the sucrose cleavage (Cho et al., 2018).

No significant variation was seen in woody tissue starch content among all the temperature treatments and scion/rootstock combinations at stage I and stage III ( Figure 9 ). This is consistent with Wanjiku and Bohne (2016) report of no significant changes in the starch content of hazelnut cultivars after low temperature treatments. Similarly, in Eucalyptus no significant variation in starch content was observed in chamber (treated at 25, 16, 13, 10, 7, 4, and 0°C for a week) and field, conditions (Leborgne et al., 1995). However, we determined the starch content in woody tissue and bark from stage II, had significant variation among scion/rootstock combinations in the control treatment ( Figures 7 , 9 ). This suggests differences in the growth rate of scion/rootstock combinations and utilization of starch among different scion/rootstock combinations.