Impaired pollen development under high-temperature conditions has been implicated in reduced yields in a large number of crop systems (Stone 2001
; Firon et al. 2006
; Prasad et al. 2006
; Mukesh et al. 2007
). In tomato, developing pollen grains are highly sensitive to HS (Pressman et al. 2002
; Firon et al. 2006
). Despite the anticipated frequency of HS problems and despite accumulated data on the high HS sensitivity of developing pollen grains, data on the mechanisms involved in pollen HSR and thermotolerance are very limited (Pressman et al. 2008
). The results presented in this study indicate that perturbation of the tomato ethylene signalling pathway, by a mutation in the ERS-like ethylene receptor, increases the sensitivity of pollen grains to MCHS conditions, as manifested by a reduction in the total number of pollen grains produced, an increase in the number of non-viable pollen grains and a significant reduction in the number of viable pollen grains in Nr
mutant plants (Table ). Previous studies analysing this mutant indicated that the Nr
mutation inhibits tomato fruit ripening, and influences fruit morphology, seed number, ascorbate accumulation, carotenoid biosynthesis, ethylene evolution and expression of many genes during fruit maturation (Alba et al. 2005
). The presented results, together with the known activity of Nr
in regulating ethylene signalling, suggest a role for ethylene and the ethylene signalling pathway in maintaining higher pollen quality under HS conditions.
We previously demonstrated that a higher quality of pollen in heat-tolerant tomato genotypes is associated with higher levels of starch accumulated during pollen maturation and higher levels of sucrose in the mature pollen grains (Firon et al. 2006
). We therefore tested the levels of starch, as well as sucrose, glucose and fructose, in maturing and mature pollen grains, respectively, of wild-type and Nr
mutant plants. Starch levels did not differ significantly between pollen of wild-type and Nr
plants (Table ). However, the Nr
mutation was shown to affect pollen carbohydrate metabolism by causing an over 2-fold reduction in the sucrose levels accumulated in mature pollen grains (Fig. ). Since starch levels did not differ between wild-type and Nr
maturing pollen, the lower sucrose levels accumulated in mature Nr
pollen grains might be due to lower sugar transport to the developing pollen and lower sucrose synthesis, rather than a direct result of starch degradation.
The significantly lower sucrose levels detected in mature pollen grains of Nr
mutant plants might contribute to their higher sensitivity to HS. High sucrose content in pollen has previously been suggested to be associated with the acquisition of germination capacity and desiccation tolerance, as it provides the necessary osmolality for cell expansion (Hoekstra and van Roekel 1988
) or is related to pollen longevity (Speranza et al. 1997
). Buitink and Leprince (2004)
suggested that sucrose participates in the formation of intracellular glass, which protects membranes during dehydration. Vesprini et al. (2002)
suggested a role for cytoplasmic pollen carbohydrates in resistance to low-temperature exposure. Indeed, we previously showed, in pepper, that both pollen quality and yield are correlated with pollen sucrose levels, being affected by different temperature regimes (with low night temperatures decreasing mature pollen sucrose levels, pollen germination capacity and yield; Pressman et al. 2006
). Kaplan et al. (2004)
pointed out a relationship between HS and cold-stress responses at the metabolite level, suggesting the possible involvement of signalling molecules and compatible solutes in temperature-stress tolerance.
It is interesting to note that exposing tomato plants to HS caused a 32-fold increase in expression of an ethylene-dependent gravitropism-deficient and yellow–green-like (ATEGY3
) gene (homologous to Arabidopsis
AT1G17870, 2E-43; Frank et al. 2009
) in developing tomato microspores. Molecular studies have revealed that a member of this gene family is involved in controlling the size and number of plastids, and encodes a metalloprotease (Guo et al. 2008
). This gene may link the HS-regulated ethylene response to pollen amyloplast biology and carbohydrate metabolism.
The involvement of ethylene in maintaining tomato pollen quality under HS is further demonstrated by: (i) the 2.6- to 10-fold increase in the number of germinating pollen grains and (ii) the significant decrease in the number of non-viable pollen grains, due to pretreatment of the plants with an ethylene releaser, prior to exposing them to STHS. This beneficial effect of ethylene pretreatment was manifested at two different stages of pollen development: 3 days before flower opening (stage A-3) and at the mature pollen stage (A), suggesting ethylene's involvement in thermotolerance of developing/maturing pollen grains as well as in pollen germination, respectively. We obtained similar results with developing pollen of Nicotiana sylvestris, another member of the Solanaceae (N. Firon et al., ARO, Israel, unpubl. res.).
Thermotolerance is generally divided into acquired (i.e. the ability to acquire tolerance to otherwise lethal HS) and basal (i.e. the inherent ability to survive temperatures above optimal growth temperatures) (Suzuki et al. 2008
and references therein). The capacity for ATT may be achieved by elevating expression levels of ‘protective’ genes prior to HS exposure (Larkindale and Vierling 2008
). The results, summarized in Fig. C and D, indicate that tomato pollen has the capacity for ATT, and that pretreating the plants with mild HS before exposing them to STHS significantly increases the number of germinating pollen grains. This suggests the activation of genes or proteins that may protect pollen germination capacity under HS.
The established ATT conditions were used to determine whether ethylene is involved in basal or acquired thermotolerance, or both, in pollen. Pretreatment of tomato plants with the ethylene biosynthesis inhibitor AVG indicated an 8.5- and 2.5-fold reduction in pollen ATT capacity at developmental stages A-3 and A, respectively, and complete inhibition of pollen germination following exposure to STHS conditions. Taken together, the results indicate involvement of ethylene in both basal and acquired thermotolerance.
The results presented in this work indicate that interfering with the ethylene signalling pathway or reducing ethylene levels increases tomato pollen sensitivity to HS, whereas increasing ethylene levels prior to HS exposure increases pollen quality. It is thus suggested that ethylene increases pollen tolerance to HS, thereby improving pollen functioning under those conditions.
In addition, involvement of ethylene in the normal course of pollen development and germination is suggested based upon the results showing a reduction in pollen quality under optimal growing conditions following pretreatment of the plants with AVG. The role of ethylene synthesis in the development and germination of tobacco pollen has previously been shown (De la Torre et al. 2006
and references therein).