- Title
- Regulation of tomato fruit set under heat stress by CWIN-mediated sucrose metabolism and signaling
- Creator
- Liu, Yong-Hua
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2015
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Emerging evidence suggests that cell wall invertase (CWIN) plays positive regulatory roles in plant responses to biotic and abiotic stresses. However, most studies have focused on vegetative stages of plant development using gene-suppression or genotypic correlation approaches and little is known about the roles of CWIN in plant early reproductive development under stress conditions. This dearth of information is particularly concerning since early reproductive development is vital for establishing crop yield potential. With global warming, heat stress is becoming a major limiting factor for crop production through aggravating fruit and seed abortion and hence irreversible yield loss. Here, we aim to determine the roles of CWIN in fruit set and development under heat stress by using a transgenic tomato in which CWIN activity is elevated through silencing its inhibitor. As sessile organisms, plants have evolved a complex heat response system to cope with higher temperature circumstances. Given the responsive mechanisms of plants may be different between severe heat stress and moderate heat stress, we evaluated the performance of CWIN-elevated transgenic tomato in fruit set and development under both short and severe heat stress (SSHS, 36/30 °C, 14 h/ 10h, imposed for 24 h) and long and moderate heat stress (LMHS, 28/20 °C, 14 h/ 10h, imposed from 10 d before pollination to 30 d after pollination). SSHS led to 100% fruit abortion in both genotypes when imposed at 0 h after pollination (hap). When imposed at 24 hap, SSHS decreased fruit growth of wild type tomato, but had no effect on that of transgenic tomato. When imposed at 48 hap, however, SSHS had no effect on either fruit set or growth in the two genotypes. Fertilization marks the transition from ovary to young fruit. Investigation of pollen tube elongation established that fertilization did not happen at 24 hap, but at 48 hap. Therefore, it appears that the ovary (24 hap) was more sensitive to SSHS than young fruit (48 hap) and the elevation of CWIN activity increased the heat tolerance of ovaries. Under LMHS, fruit set of wild type tomato was decreased by 66% compared to that under control conditions. By contrast, transgenic tomato showed no reduction in fruit set, indicating that elevated CWIN activity improves fruit set and growth under LMHS and SSHS, respectively. Fruit set determines the final fruit number and has a greater influence on final fruit yield than fruit size. As sumarrized above, SSHS imposed at 0 hap led to 100% fruit abortion in both genotypes, redering it impractical to study impact of CWIN on fruit set under this condition. However, under LMHS, CWIN-elevated transgenic tomato showed higher fruit set than wild type tomato. Moreover, moderate heat stress is a more frequent event than severe heat stress in the field. Therefore, further studies were performed to examine the roles of CWIN in tomato fruit set under LMHS, focusing on 2 d before pollination (dbp) ovaries and 2 d after pollination (dap) fruits, which are the key period for fruit set. LMHS did not reduce photosynthesis and plant biomass in either genotype. However, the rate of sucrose (Suc) import into 2 dap fruit was reduced in wild type tomato under LMHS, but remained unchanged in CWIN-elevated transgenic tomato. The observation indicates increased sink strength in the CWIN-elevated tomato fruit may contribute to the improved fruit set under LMHS. Further studies showed that LMHS increased the activities of hexokinase (HXK) and fructokinase (FRK) in transgenic ovary/fruit, but had no effect on those in wild type tomato, suggesting an increased hexose utilization in transgenic tomato under LMHS. A new, but more stable sugar homeostasis was also established in transgenic tomato under LMHS. Here, the Glc and Fru content in transgenic 2 dbp ovaries remained unchanged under LMHS, but decreased in wild type ovaries. In addition, the Suc and starch content increased under LMHS in wild type 2 dbp ovaries and 2 dap fruit, respectively, but remained unchanged in transgenic tomato. Inhibited cell division and/or induced programmed cell death (PCD) are the two major factors responsible for fruit and seed abortion under heat stress. We postulated that the increased hexose utilization and more stable sugar homeostasis in transgenic tomato may enhance fruit set under LMHS through promoting cell division and/or inhibiting PCD. Surprisingly, no genotypic difference was observed between wild type and transgenic tomato in cell number and size of ovary/fruit under LMHS. Investigation into PCD in 2 dap fruit revealed that PCD was dramatically induced in seeds and pericarp of wild type fruit under LMHS. However, only a weak and no PCD signal was detected in transgenic seeds and pericarp, respectively, based on TUNEL assay. The findings indicate that elevation of CWIN activity blocked or delayed PCD without affecting cell division in tomato ovaries and fruits. Reactive oxygen species (ROS)-derived oxidative stress has been well recognized as the important trigger in heat stress-induced PCD. Thus, several physiological markers of oxidative stress response were examined, including the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), and the content of H₂O₂ and malondialdehyde (MDA). However, no difference was observed between the two genotypes in these parameters under LMHS. Thus, the overall conclusion is that elevated CWIN activity in transgenic tomato enhances fruit set under LMHS possibly through inhibiting PCD in a ROS-independent manner without affecting cell proliferation. To explore the molecular basis for these findings, the transcripts of genes encoding cell cycle-related proteins (cyclins and cyclin-dependent kinase), PCD-related proteins and antioxidant enzymes were measured using the same set of ovary and fruit samples harvested under LMHS. There was no difference in mRNA levels of cell cycle-related genes including two cyclin-dependent kinase genes (CDKA1 and CDKB1;1) and three cyclin genes (CycA2;1, CycB2;1 and CycD3;1) between wild type and transgenic tomato. On the other hand, however, compared to wild type tomato, transgenic tomato had lower transcript levels of PCD-related genes in 2 dap fruit under LMHS, including the nuclease gene TBN1, cysteine protease gene RD19α serine protease gene SBT3 and the cupin-domain protein gene pirin. At the same time, little genotypic difference was observed in transcript levels of antioxidant enzyme genes (SOD1, SOD2, APX1, APX2, CAT1 and CAT2) under LMHS. Thus, the results of this expression analysis are consistent with findings at cellular and physiological levels. Noticeably, different from LMHS, SSHS increased the content of H₂O₂ and MDA in both genotypes. At the same time, SSHS increased the expression of SOD2, APX2 and CAT2 in wild type 2 dbp ovaries. However, SSHS only increased the expression of CAT2 in transgenic tomato. These data indicate that CWIN regulates SSHS and LMHS responses of tomato fruit in an oxidative-stress dependent and independent manner, respectively. Heat stress transcription factors (Hsfs) and heat shock proteins (Hsps) play positive roles in plant heat tolerance. Compared to wild type tomato, transcript levels of Hsf genes (HsfA1, HsfA2 and HsfB1) and Hsp genes (Hsp90, Hsp100 and HspII17.6) were enhanced in transgenic tomato under LMHS. Thus, the increased hexose utilization in CWIN-elevated transgenic tomato, as reflected by increased activity and expression of HXK and FRK in transgenic tomato, may enhance tomato fruit set under LMHS through fuelling the expression and biosynthesis of Hsfs and Hsps, which is energy- and metabolite- consuming. Auxin is required for successful fruit set and development and positive correlations have been found between HXK-mediated sugar and auxin signaling pathways. With increased HXK activity and expression in transgenic 2 dbp ovaries, the transcript level of IAA9, a negative regulator of auxin signaling, was lower in transgenic ovary/fruit than wild type under LMHS. On the other hand, the expression levels of two auxin biosynthesis genes ToFZY1 and ToFZY6 were enhanced in transgenic ovary/fruit under LMHS as compared to wild type tomato. G-protein-mediated signaling pathaway interacts with auxin signaling pathway. As a potential sensor for extracellular sugars, regulator of G-protein signaling (RGS) may also play regulatory roles in auxin signaling through negatively modulating G-protein signaling. Indeed, the transcript level of RGS1 was up-regulated in wild type 2 dap fruits under LMHS, but not in transgenic fruits. Therefore, the elevated CWIN activity in transgenic fruit appears to enhance the biosynthesis and signaling of auxin and hence fruit set under LMHS likely via HXK- and RGS-mediated sugar signaling. Overall, the elevation of CWIN activity by silencing its inhibitor increases tomato fruit set under LMHS possibly through inhibiting PCD in a ROS-independent manner without affecting cell proliferation. The increased heat tolerance in CWIN-elevated transgenic tomato may be related to enhanced Suc import into, and subsequent hexose utilization in, ovary/fruit under LMHS, which appears to enhance fruit set through fuelling the biosynthesis of Hsfs and Hsps and promoting auxin signaling and biosynthesis.
- Subject
- tomato; fruit set; cell wall invertase; sucrose metabolism; sugar signaling; heat stress
- Identifier
- http://hdl.handle.net/1959.13/1310448
- Identifier
- uon:22036
- Rights
- Copyright 2015 Yong-Hua Liu
- Language
- eng
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