Impact of temperature and cultivar on chlorophyll fluorescence parameters
It is evident that fluorescence Fo is low in case of unstressed plants and increases in case of stressed plants. Fm is admitted to be low in stressed plants. Between the two fluorescence limits (Fo and Fm), we can deduce the quantum efficiency component that is always less than 1. Quantum yield decreases when the rate of inhibitors increases. For unstressed leaves, the value of Fv/Fm is about 0.83.
Table 1 shows the impact of temperature and cultivar on various chlorophyll fluorescence parameters.
The impact of temperature on various chlorophyll fluorescence parameters was analyzed first during the stolon initiation stage (24 DAP), during this stage and on average, plants cultivated at low temperature demonstrated the lowest estimation of Fo (266.72), which was significantly different (P≤0.01) compared to intermediate (290.61) and high temperature (296.94). This indicates that plants cropped in low temperature regime (18/16°C, day/night) seem to be less stressed compared with other temperature conditions. During 49 DAP, the lowest average value of Fo was that of intermediate temperature (368.08), followed by the low temperature condition with (304.66), during this growth stage the low temperature showed also the highest value of Fm with 1513.00 (Table 1). At 64 and 78 DAP, on average, the intermediate temperature condition demonstrated the highest estimation of Fm (Table 1).
Those results showed that during the stolon initiation stage, plants cultivated at low temperature performed better concerning photosynthesis compared to intermediate and high temperature. Later, from 49 DAP, plants cultivated at intermediate temperature showed higher photosynthesis efficiency.
During tuber initiation stage, there was a significant cultivar*temperature interaction for Fo with P≤0.01. Data demonstrate that for all studied temperatures, Bellini showed highest values of Fo (Table 1). Analysis shows also a significant cultivar*temperature interaction for Fm. Highest values of Fm were those of Bellini cultivar, especially at high temperature. Those results indicate that Spunta performed better concerning photosynthesis effeciency compared to Bellini. We also observed a significant cultivar*temperature interaction at the level of 5% for yield of photosynthesis. Spunta behaved better at high and low temperatures, showing a Fv/Fm of about 0.83 in both regimes, and about 0.80 at intermediate temperature (Table 1).
Table 1 shows a significant cultivar*temperature interaction (P≤0.01) for Fo at 30 DAP. Bellini registered the highest Fo in all temperatures compared to Spunta.
During the tuber bulking phase (49 DAP) the chlorophyll fluorescence parameters were not significantly impacted by temperature. Later, during the end of the tuber bulking stage 64 DAP, the interaction between cultivar and temperature was statistically significant for Fm. For this trait, the Spunta cultivar had the highest level in all temperature conditions. Spunta showed the lowest Fv/Fm ratio, regardless of the temperature condition. Also at tuber maturation 78 DAP, no significant effect of temperature regimes was shown for chlorophyll fluorescence parameters (Table 1).
All together these results show that photosynthetic efficiency was higher for Spunta cultivar during different growth stages and that the two cultivars behaved differently according to the temperature condition.
Impact of temperature on soluble sugars and starch in leaves, stems and tubers
In order to elucidate temperature responses of potato plants, photosynthetic leaves were analyzed with regard to their carbohydrate contents (glucose, fructose, sucrose and starch) first during tuber initiation stage (30 DAP). Temperature significantly affects hexose and starch accumulation in leaves during this stage. Plants cultivated at low temperature showed maximal amounts of glucose (0.188 µg/mg FW), followed by high temperature (0.157 µg/mg FW), which were significantly different (P≤0.01) compared to glucose level in leaves at intermediate temperature (0.041 µg/mg FW) (Fig. 1A). Highest fructose level (Fig. 1B) was noted in the high temperature regime (0.240 µg/mg FW), followed by low temperature (0.218 µg/mg FW). Those values were significantly different compared to fructose content in leaves at intermediate temperature (0.070 µg/mg FW). Sucrose content (Fig. 1C) was higher at low temperature (0.184 µg/mg FW), followed by high temperature (0.159 µg/mg FW), lowest sucrose accumulation was recorded at intermediate temperature. Starch accumulation in leaves (Fig. 1D) was higher under low temperature conditions (0.180 µg/mg FW), followed by high temperature (0.179 µg/mg FW). Lowest level of starch accumulation was obtained at intermediate temperature (0.033 µg/mg FW).In the leaf tissue representing the source organ, the content of soluble sugars was not significantly affected by the different temperature regimes during the tuber bulking stage, 49 DAP (Table 2).
However, during the end of the tuber bulking stage (64 DAP), sugar determination as presented in Table 2, revealed significant temperature effects on the amount of glucose accumulated in source leaves. During this growth stage, highest amount of glucose was recorded in the low temperature condition (0.219 µg/mg FW), followed by the elevated temperature condition with 0.127 µg/mg FW. Lowest levels of glucose were found in leaves taken from plants cultivated at intermediate temperature (0.064 µg/mg FW). Also with regard to fructose content, the highest amount was detected under low temperature conditions (0.387 µg/mg FW), which was statistically different from high temperature (0.197 µg/mg FW) and intermediate temperature (0.127 µg/mg FW) conditions.
Also during the tuber maturation phase (78 DAP) the amount of glucose accumulated in leaves strongly depends on the applied temperature (Table 2, level of 5%). Higher amounts of glucose were observed under high temperature conditions (0.189 µg/mg FW), which was statistically different compared to low temperature conditions (0.110 µg/mg FW). The value was lowest under intermediate temperature conditions (0.069 µg/mg FW). Table 2 shows that at high temperature also the fructose content was highest (0.238 µg/mg FW), which was statistically different compared to low temperature (0.153 µg/mg FW).
The highest level of glucose in the stem at 49 DAP was recorded at low temperature with 1.549 µg/mg FW, followed by the high temperature regime with 1.369 µg/mg FW, both of which were significantly different compared to plants cultivated in the intermediate temperature condition with 0.359 µg/mg FW (Table 2).
Table 2 also shows that the analysis of variance (ANOVA) revealed a significant impact of temperature on fructose in the stem (P≤0.05). High and low temperature conditions displayed the highest levels of fructose in stems with respectively 0.214 and 0.187 µg/mg FW. The lowest level of fructose was measured at intermediate temperature (0.053 µg/mg FW). Table 2 shows a significant impact of temperature on sucrose content in the stem (P≤0.01) during the end of the tuber bulking stage (64 DAP). Maximum sucrose content was measured in low and high temperature regimes with respectively 0.064 and 0.056 µg/mg FW. At intermediate temperature, a substantial decrease in sucrose content in the stem tissue was measured (0.027 µg/mg FW).
In the stem tissue predominantly representing transport phloem, glucose content was not significantly affected by the different temperature regimes during the tuber maturation stage (Table 2).
Regarding the sink organ, soluble sugars content was not significantly affected by the different temperature regimes during the tuber bulking stage (Table 2).
With regard to starch content in tubers, contrary to the accumulation of hexoses, the highest value was recorded in the intermediate temperature condition with 0.683 µg/mg FW in the end of the tuber bulking stage, followed by high and low temperature conditions with 0.667 and 0.646 µg/mg FW respectively (Table 2).
In sink organs however, namely in potato tubers, glucose content differed significantly according to the temperature during the tuber maturation stage with the highest amount (0.155 µg/ mg FW) at elevated temperature (Table 2). The same holds true for fructose content in tubers during the maturation phase (with 0.017 µg/mg FW at high temperature).
Also tuber starch content was highest at high temperature with 1.349 µg/mg FW, and lowest at intermediate temperature with 0.968 µg/mg FW.
Impact of temperature on enzymes activities
Analysis of variance (ANOVA) of acid invertase activity in stems (Fig. 2A) at 49 DAP revealed a significant effect of temperature on the activity of this enzyme (P≤0.05). The highest activity was detected at low and intermediate temperature with 6.881 and 6.854 nmol*min-1*mg protein-1 respectively.
There were no significant enzyme activities during the end of the tuber bulking stage (64 DAP).
The activity of SPS, the key enzyme of sucrose synthesis, was determined in leaves at 78 DAP (tuber maturation stage) (Fig. 2).
Surprisingly, the highest SPS activity was detected at low temperature with 9.153 nmol*min-1*mg protein-1 (Fig. 2). Intermediate and high temperature showed very similar activities with 7.191 and 7.806 nmol*min-1*mg protein-1 respectively.
There was a significant interaction between cultivar andtemperature for SPS activity (Fig. 2B). While SPS activity in the Spunta cultivar was not affected by temperature changes, the Bellini cultivar showed the highest SPS activity under low temperature conditions (Fig. 2B).
Thus, sucrose synthesis via SPS as well as sucrose cleavage by acid invertases both are highest under low temperarure.
Impact of temperature and cultivar on the expression patterns of genes related to starch and sucrose metabolizing enzymes
In order to identify the expression profiles of genes related to starch and sucrose metabolisms and analyze their roles in the tuberization process, transcript levels of three genes were surveyed using quantitative real-time PCR (qPCR) technology (Fig. 3). These three genes showed different expression patterns depending on cultivar and temperature during the tuber initiation stage (30 DAP) (Fig.3A).
We analyzed the expression level of the large subunit of AGPase, which was previously shown to physically interact with the major potato phloem loading sucrose transporter StSUT1(Krügel et al., 2012).
In leaves, the expression level of the AGPase LSU was relatively high in all temperature regimes. Even the Spunta cultivar showed a high expression of AGPase at high and low temperature, while, in the intermediate temperature condition, Bellini gave the highest AGPase transcript amount. The expression level of the cell-wall invertase (StInv6) in potato leaves was relatively low under all temperature conditions in both cultivars, except in Spunta growing at the high temperature regime, suggesting the more active role of StInv6 in sucrose conversion in source organs at high temperature (Fig. 3A). At high and low temperatures, the expression level of the SuSy gene StSUS4 remained low compared to AGPase and StInv6 expression with Bellini showing higher transcript levels than Spunta. At intermediate temperature, StSUS4 expression reached the highest levels.
The expression pattern of genes related to starch and sucrose metabolism during tuber bulking phase is shown in Fig. 3B.
Expression of AGPase LSU in Spunta leaves was highest under low temperature conditions, whereas Bellini showed highest levels at intermediate temperature. At high temperature, both cultivars displayed a relatively low level of StInv6 expression, which is consistent with the low invertase activity shown in Fig. 3B. StSUS4 transcript amount is highest at high temperature in both cultivars (Fig. 3B).
The gene expression analysis via qPCR revealed that in the end of the tuber bulking phase the AGPase expression is again highest under low temperature conditions (Fig. 3C), also suggesting that starch build up in leaves is highest under these conditions.
Also the expression of sucrose and starch metabolizing enzymes was assessed during the tuber maturation phase (78 DAP) in leaves (Fig. 3D). While the StLin6 transcript amount was highest at intermediate temperature in both cultivars, the Bellini cultivar showed high StSUS4 transcript amounts regardless of the temperature (Fig. 3D), suggesting that the sucrose synthase pathway converting sucrose into starch dominates the invertase pathway at this developmental stage in Bellini.
The detailed expression analysis of sugar metabolizing enzymes is not necessarily reflected by the activity measurements shown in Fig. 3. Whereas invertase activity in leaves is highest under low temperature, no such correlation could be demonstrated regarding the transcript level of invertase StLin6.
Impact of temperature and cultivar on production parameters
Determination of the total tuber yield per plant at 64 DAP (Fig. 4) revealed a significant impact of temperature on this trait. The highest tuber yield was produced at low temperature with 118.5 g, followed by high temperature with 114.6 g per plant. Tuber yield decreased at intermediate temperature (75 g).
For the number of tubers per plant (Fig. 4B), analysis of variance ANOVA revealed significant differences between the tested temperature conditions.
Elevated temperature brought about the highest number of tubers with 7.7, followed by low temperature with 7 tubers per plant. The number of tubers decreased in the intermediate condition.
An impact of the temperature on both the total tuber yield and the tuber number per plant was observed. While higher temperature favored total tuber yield, tuber number seemed to be higher with decreasing temperature.
There was a significant cultivar-by-temperature interaction for these production parameters. The Bellini cultivar produced a higher tuber yield compared to Spunta, regardless of the temperature conditions (Fig. 4). Also the number of tubers per plant was generally higher for Bellini than for Spunta when tubers were harvested during the end of the tuber bulking phase (64 DAP).
The production parameters were determined also during the tuber maturation phase (78 DAP, Fig. 5) with significant differences according to the growing temperature. Interestingly, the highest tuber yield was recorded at high temperature, whereas at intermediate temperature, which was assumed to be optimal for tuber production, the lowest yield was recorded for both cultivars at this stage (Fig. 5). The highest number of tubers per plant was observed under low temperature conditions (Fig. 5B). The Bellini cultivar generally produced more tubers and a higher total tuber yield than Spunta, irrespective of the temperature (Fig. 5C, D).
Final yield (90 DAP)
The final tuber yield was determined at 90 DAP (Fig. 6). Here again, the temperature had a significant impact on this trait (P≤0.01). For both cultivars, the highest tuber yield was recorded at high temperature with 150.8 g, followed by low temperature with 136 g. The lowest yield was maintained at intermediate temperature (119.876 g). Regarding the number of tubers per plant, low temperature conditions seem to be most favorable for both cultivars (Fig. 6). The temperature impact shows significant differences (P≤0.01). Also at 90 DAP, the Bellini cultivar produced the highest total yield and the highest tuber number compared to Spunta (Fig. 6C, D).
A correlation study was performed with most of the measured parameters affecting tuber physiology (Table 3).
There was a strong positive correlation among glucose, fructose, sucrose and starch contents in leaves.
A positive correlation was also found between fructose and starch contents in sink organs: fructose and starch levels increase in parallel in potato tubers. Acid invertase activity in the stem was shown to correlate with fructose and starch levels in tubers. This correlation can be interpreted as high sucrose cleavage activity after phloem unloading having a positive effect on fructose and starch accumulation in sink organs.
Acid invertase activity in the stem negatively correlates with sucrose and starch accumulation in leaves. Presumably, high sucrose cleavage activity via acid invertases in the stem tissue could increase sink strength and thereby promote sucrose export from leaves and conversion of transitory starch in leaves into the transport form sucrose.
There was a positive correlation between SPS activity in leaves and glucose and starch accumulation in stems.
A strong positive correlation was shown between acid and neutral invertase activities in tubers.
Acid invertase activity in stems is correlated with the expression level of the invertase gene (StInv6) in leaves.
A positive correlation was also found between final weight and number of tubers per plant.
Most interestingly, final tuber yield as well as tuber number per plant both correlated with the expression level of AGPase and StSUS4 genes in leaves. This indicates a strong impact of sucrose and starch metabolism in source leaves on both tuber initiation and tuber development, both affecting final tuber number and tuber yield.