High invertase activity in tomato reproductive organs correlates with enhanced sucrose import into, and heat tolerance of, young fruit

Abstract

Heat stress can cause severe crop yield losses by impairing reproductive development. However, the underlying mechanisms are poorly understood. We examined patterns of carbon allocation and activities of sucrose cleavage enzymes in heat-tolerant (HT) and -sensitive (HS) tomato (Solanum lycopersicum L.) lines subjected to normal (control) and heat stress temperatures. At the control temperature of 25/20 °C (day/night) the HT line exhibited higher cell wall invertase (CWIN) activity in flowers and young fruits and partitioned more sucrose to fruits but less to vegetative tissues as compared to the HS line, independent of leaf photosynthetic capacity. Upon 2-, 4-, or 24-h exposure to day or night temperatures of 5 °C or more above 25/20 °C, cell wall (CWIN) and vacuolar invertases (VIN), but not sucrose synthase (SuSy), activities in young fruit of the HT line were significantly higher than those of the HS line. The HT line had a higher level of transcript of a CWIN gene, Lin7, in 5-day fruit than the HS line under control and heat stress temperatures. Interestingly, heat induced transcription of an invertase inhibitor gene, INVINH1, but reduced its protein abundance. Transcript levels of LePLDa1, encoding phospholipase D, which degrades cell membranes, was less in the HT line than in the HS line after exposure to heat stress. The data indicate that high invertase activity of, and increased sucrose import into, young tomato fruit could contribute to their heat tolerance through increasing sink strength and sugar signalling activities, possibly regulating a programmed cell death pathway.

Fig. 1.

Comparison of flower (A) and fruit (B) numbers and fruit-set percentages (C) between field-grown HT and HS lines. Fruits from trusses 1–3 were set in June 2009 with a daily mean temperature of 26 °C (moderate temperature), while fruits from trusses 4–6 were set in July with daily mean temperature of 29 °C (high temperature). Each value is the mean ± SE from 10 biological replicates. An asterisk indicates a significant difference between the two lines at a given truss position (Student’s t test, *P < 0.05; **P < 0.01, ***P < 0.001).

Fig. 2.

Rates of sucrose (suc) import into young fruits and the corresponding fruit biomass gain. (A) Sucrose import rates from pedicels into 5–6-day fruits of HT and HS lines. (B) Average dry-weight gains of 5–6-day fruits of HT and HS lines. (C) Five-day fruit fresh and dry weights. Each value is the mean ± SE from 10 biological replicates. An asterisk indicates a significant difference (Student’s t test, *P < 0.05; **P < 0.01).

Fig. 3.

Whole-plant biomass partitioning in HT and HS lines. Fresh and dry (biomass) weights were measured for fruits (A), stems plus leaves (B), and roots (C) at fruit maturity from HT and HS lines. (D) Whole-plant weight from HT and HS lines. (E) Percentage of biomass in fruit and vegetative organs at maturity. Each value is the mean ± SE from eight biological replicates. An asterisk indicates a significant difference (Student’s t test, *P < 0.05; **P < 0.01).

Fig. 4.

INV and SuSy activities of 5-day fruit from HT and HS lines at the control temperature or after mild heat stress (30/25 °C, day/night). (A) INV and SuSy activity of 5-day fruit from HT and HS lines at the control temperature of 25/20 °C (day/night). (B) INV and SuSy activity of 5-day fruit from HT and HS lines after 30 °C treatment for 2 h during daytime. (C) INV and SuSy activity of 5-day fruit from HT and HS lines after 25 °C treatment for 4 h at night. (D) INV and SuSy activity of 5-day fruit from HT and HS lines after 30/25 °C (day/night) mild heat stress for 24 h. (E, F) Pattern of INV and SuSy activity of 5-day fruit of the (E) HT line and (F) HS line exposed to the control temperature (25/20 °C, day/night) compared with a 2-h short-term mild heat stress (30 °C, daytime) immediately before sampling fruit. (G, H) Pattern of INV and SuSy activity of 5-day fruit of the (G) HT line and (H) HS line exposed to the control temperature (25/20 °C, day/night) compared with a 24-h long-term mild heat stress (30/25 °C, day/night) immediately before sampling fruit. Each value is the mean ± SE from three biological replicates. An asterisk indicates a significant difference (Student’s t test, *P < 0.05; **P < 0.01).

Fig. 5.

INV and SuSy activities of flowers and 5-day fruits from HT and HS lines at the control temperature or after 24 h of strong heat stress (36/28 °C, day/night). (A) INV and SuSy activity of 5-day fruit from HT and HS lines after 24 h of strong heat stress (36/28 °C, day/night). (B) INV and SuSy activity of 0-day flower from HT and HS lines at the control temperature of 25/20 °C (day/night). (C) INV and SuSy activity of 0-day flower from HT and HS lines after 24 h of strong heat stress (36/28 °C, day/night). Each value is the mean ± SE from three biological replicates. An asterisk indicates a significant difference (Student’s t test, *P < 0.05; **P < 0.01).

Fig. 6.

RT-PCR analysis of the expression profiles of INV genes from 5-day fruits of HT and HS lines at the control temperature (25/20 °C, day/night) and after 24-h mild (30/25 °C, day/night) or strong (36/28 °C, day/night) heat stress. For mild heat stress, all genes were amplified by 30 cycles. For strong heat stress, Lin5 and TIV1 were amplified by 25 cycles; Lin6, Lin7, and INVINH1 were amplified by 30 cycles. The reference gene, Actin, was amplified by 28 cycles.

Fig. 7.

Western blot analyses of 5-day fruit from HT and HS lines at the control temperature (25/20 °C, day/night) and after 24-h mild heat stress (30/25 °C, day/night). (A) Protein gel blot analysis of CWIN, Lin5. Arrowhead indicates position of Lin5 at 64 kDa. (B) Protein gel blot analysis of invertase inhibitor, INVINH1. Note, a band at the predicted size of 16.4 kDa for INVINH1 (arrowhead) was evident at the control temperature for both lines but became hardly detectable at mild heat stress. (C) Coomassie blue stain of SDS/PAGE gel of cell wall protein of 5-day fruit from the HT and HS lines. The far-right lane is a molecular-mass-marker lane.

Fig. 8.

RT-PCR analysis of expression levels of two sHSP genes and one senescence gene (LePLDa1) of 5-day fruit from HT and HS lines exposed to the control temperature (25/20 °C, day/night) and after 24-h strong heat stress (36/28 °C, day/night). The sHSP genes, LeHsp 17.4-CII and LeHsp 17.6-CII, were amplified by 30 cycles and the senescence gene, LePLDa1, was amplified by 25 cycles. The reference gene, Actin, was amplified at 30 cycles.