Impacts of heatwaves and drought on foliar N-containing metabolites
Of the 20 common proteinogenic AAs, 18 were quantifiable by our HPLC system. In addition, we quantified γ-aminobutyric acid (GABA) and ornithine (Orn), two of the common non-protein AAs in plants (Fig. 1, 2, S1, S2). Before the first heatwave event in 2016, AA concentrations were similar among the treatment groups apart from Glu and Gln in spruce and Val in birch (Fig. 1, 2, S1, S2). The response of the spruce plants to the combined heat+drought stress was vastly different from the response of the birch (Fig 1, 2). The HD spruce displayed several novel changes in the concentration of 10 AAs (Gln, Ser, Pro, GABA, Val, Ile, Trp, Phe, Cys, and His) in August at the end of the 2016 season (Fig. 1). There were no statistically significant differences in AAs between the H plants and the C plants, or the D plants and the C plants in late August 2016. By the start of the 2017 season, all previously elevated AAs had reverted back to levels similar to the C plants, with the exclusion of Asp (increased inHD plants) and Glu and Val (decreased in HD plants; Fig. 1a, b, h). By the end of the second summer of heatwave stress, theHD spruce tended to respond similar to the D plants (Asp, Ala, His; Fig. 1a, e, o) or the response was between that of theD and H response (Phe, Orn; Fig. 1l, n). There were a few statistically significant differences found between the D ,H , and HD spruce and the C plants after two years of repeated stress (Glu, H vs C ; Ala, HD vsC ; and Orn, D vs C ).
The birch plants did not exhibit the same extreme responses of AA metabolism to the HD treatment as the spruce. At the end of the first season of stress, Phe and Trp were the only AA to accumulate under the combined stress (Fig. 2j, k; Phe, p < 0.05HD vs C ; Trp, p < 0.05 HD vsC ). Compared to the H treatment, Cys was reduced in theD and HD treatments at this time as well (Fig. 2k, l). At the start of the second season, only few differences in AAs were found among treatment groups and none were unique to the HD treatment (Ser, Leu, Orn; Fig. 2b, i, m). By the end of the second summer of heatwave stress, unlike the spruce plants, the HD birch did not display unique responses from either independent stress. Instead theHD birch tended to respond more similarly to the D plants than the H plants in several AAs (Ala, Pro, GABA, Val, Ile, Orn; Fig. 2d, e, f, g, h, m).
Of the three PAs quantified, putrescine (Put) was the most abundant in spruce tissue whereas spermidine (Spd) or spermine (Spm) was the most abundant in the birch (Table 1). Prior to the first heatwave in 2016, no differences were found among treatment groups of either species. At the end of the 2016 season, the HD spruce produced significantly less Put (p < 0.05 HD vs C ) and Spd (p< 0.05 HD vs C ) than the C plants where concentrations of these PAs were < 50% of that of theC plants (Table 1). Overall, Put concentrations were higher in the spruce foliage in the second year compared to the first year. In the beginning of the second season, all treatment groups of spruce had similar foliar PA concentrations. A decrease in Put and Spd was observed in the HD spruce at the end of each season. Although PA quantities in the D and H spruce were not impacted by one season of stress, two seasons of recurrent stress did result in decreased PA metabolism. In late August of 2017, the D, H,and HD spruce had lower concentrations of Put (Table 1; p < 0.01 D vs C ; p < 0.01H vs C; p < 0.01 HD vs C ) and Spd than the C plants (Table 1; p < 0.01 D vsC ; p < 0.001 H vs C; p <0.01 HD vs C ). At this time, the H and HDplants also had higher concentrations of Spm compared to the Cplants (p < 0.001 H vs C; p <0.05 HD vs C ).
In birch, the greater quantities of Spd and Spm where found in the first year. Very few changes in foliar PA concentration were found in the stress-treated birch across both years (Table 1). By the end of the first year, differences among treatment groups were only found in Spd where H plants had a significantly higher concentration than theC , D , and HD plants, and the D and HDplants concentrations were 74% lower than the C plants (Table 1;p < 0.01 D vs C ; p < 0.01 HD vs C ). For birch, the second year of treatments did not result in statistical differences among treatment groups at the beginning or end of this season except for higher Spm concentration in the H -treated plants than the D and HD plants (Table 1; p < 0.05 H vs D ; p < 0.05 H vsHD ).
In June 2016, no differences were found in total soluble protein (TSP) among treatment groups for either species. The unique response observed in the HD spruce in TSP and total chlorophyll concentration in late August 2016 was analogous to the response of several AAs at this time. The HD spruce produced significantly more TSP than theC , D , and H treatments where these plants produced 140% more TSP than the C plants (Fig. 3a, S3a; p< 0.001 HD vs C , D , H ). Chlorophyll a +b concentration decline by nearly 70% in the HD plants as well (Fig. 3b; p < 0.001HD vs C ). By the following season, TSP and Chlorophyll a+b content in the HD spruce no longer differed from theC plants. At the end of the first season, no statistical differences in total leaf N or C were found among any of the treatment groups in spruce (Fig. 3, S3). At the start of the second year, the only significant differences among treatment groups were found in C content where the H and HD plants 3-4% more total C than theC plants (Fig. 3d, S3d). By the end of the second season of recurrent stress, only the -treated spruce exhibited a reduction in total leaf N by >30% compared to the Cplants (p < 0.05 D vs C ).
Total soluble protein in birch was not affected by treatment at any of the sampling dates (Fig. 3e, S3e). Unlike the unique response in chlorophyll a +b observed in spruce after one season ofHD stress, all birch treatment groups had 28-33% less foliar chlorophyll a +b than the C plants (Fig. 3f S3f;p < 0.05 D vs C ; p < 0.05 H vs C ; p < 0.05 HD vsC ), but total N and C in these plants was not affected (Fig. 3g, h, S3g, S3h). At the start of the second season, all treatments had similar TSP, Chlorophyll a +b , total N, and total C. By the end of year two, only total leaf N differed among treatment groups where the D and HD birch had lower N than the H plants (Fig. 3g, S3g; p < 0.05 D vs H ; p< 0.05 HD vs H ).