*5.2. Soluble Sugars*

Soluble sugars are necessary for pollen viability and germination. An imbalance in the sugar metabolism caused by HS is associated with the failure of tomato plant fruit set [29]. When developing tomato anthers are continuously exposed to HT, the carbohydrate metabolism is altered, resulting in the reduction of the number of pollen grains per flower and pollen viability [67]. Heat-tolerant tomato genotypes have a higher carbohydrate concentration in pollen grains than susceptible ones under HS [21]. In addition, heattolerant tomato genotypes accumulate more soluble sugars in their leaves under HS than susceptible ones at the flowering and anthesis stages [39], possibly due to their better performance in maintaining carbohydrate synthesis under HS.

#### *5.3. Proline*

The development and fertility of pollens depend on local proline biosynthesis in mature pollen grains, as well as during the later microspore development stages [83]. Proline also functions as a molecular chaperone, regulating the protein structure and protecting cell damage in stress conditions [84,85]. In tomato plants, the disruption of proline transport to the anther may be a possible cause of the reduction in pollen viability [29]. Proline accumulation in pollens is affected more significantly in heat-susceptible tomato cultivars than in heat-tolerant ones [38]. The proline content in leaves can be a useful measure of stress in tomato plants [86]. Changes in proline content under HT, as well as the endogenous level of proline content, differ according to genotypes [87]. Seedlings of a heat-tolerant cultivar accumulate significantly less proline in leaves than those of a susceptible one in HT [36]. The proline content of a tolerant cultivar did not show significant change but that of a susceptible one showed a continuous increase during the HT treatment period [36].

To understand the relationship between proline content and HT tolerance in tomatoes and to test the possibility of using proline content in heat tolerance screening, we grew 43 tomato genotypes in greenhouses where the temperature set-point for ventilation was 28 ◦C and 40 ◦C for CK and MCHS, respectively. The proline content, pollen germination, pollen tube length, and fruit set and yield of 43 genotypes were investigated; correlation analyses were conducted according to Sherzod et al. [56]. In CK, the proline content in leaves was significantly correlated with pollen germination (*r* = 0.377 \*) and fruit set (*r* = 0.415 \*\*) (Table 1) but no significant correlation was observed between proline content and other traits in MCHS (Table 2). The significant correlation between pollen germination and fruit set in CK but not in MCHS may be due to differences in genotype-dependent proline accumulation in leaves [36], resulting in disrupted proline transport in HT [29]. The results indicate that the use of proline content in leaves for heat tolerance screening is still premature and further study is necessary.

**Table 1.** Correlation between biochemical and reproductive traits at control temperatures (28 ◦C).


\* and \*\* indicate significant difference at *p* < 0.05 and *p* < 0.01 levels, respectively.

**Table 2.** Correlation between biochemical and reproductive traits at high temperatures (40 ◦C).


\* and \*\* indicate significant difference at *p* < 0.05 and *p* < 0.01 levels, respectively.

#### *5.4. Glycine Betaine*

Glycine betaine is a compatible osmolyte and plays an important role in osmoregulation in plants. It is synthesized in both chloroplasts and cytoplasm, but only glycine betaine in chloroplasts is positively related to stress tolerance [88]. This implies that high glycine betaine content may not necessarily account for enhanced stress tolerance. In tomato plants, however, glycine betaine significantly increased in heat-stressed tomato plants, in comparison with non-stressed plants [89]. The exogenous application of glycine betaine to heat-stressed tomato plants enhanced seed germination, the expression of heat-shock genes, and the accumulation of heat-shock protein 70 [90] and fruit yield in open fields [91].
