Cell Wall Invertase Promotes Fruit Set under Heat Stress by Suppressing ROS-Independent Cell Death 1[OPEN] Yong-Hua Liu 2 , Christina E. Offler, and Yong-Ling Ruan* School of Environmental and Life Sciences and Australia-China Research Centre for Crop Science, The University of Newcastle, Callaghan, NSW, 2308, Australia (Y.-H.L., C.E.O., Y.-L.R.); and Institute of Vegetable Science, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China (Y.-H.L.) ORCID IDs: 0000-0001-8545-9192 (Y.-H.L.); 0000-0002-5403-4834 (C.E.O.); 0000-0002-8394-4474 (Y.-L.R.). Reduced cell wall invertase (CWIN) activity has been shown to be associated with poor seed and fruit set under abiotic stress. Here, we examined whether genetically increasing native CWIN activity would sustain fruit set under long-term moderate heat stress (LMHS), an important factor limiting crop production, by using transgenic tomato (Solanum lycopersicum) with its CWIN inhibitor gene silenced and focusing on ovaries and fruits at 2 d before and after pollination, respectively. We found that the increase of CWIN activity suppressed LMHS-induced programmed cell death in fruits. Surprisingly, measurement of the contents of H 2 O 2 and malondialdehyde and the activities of a cohort of antioxidant enzymes revealed that the CWIN- mediated inhibition on programmed cell death is exerted in a reactive oxygen species-independent manner. Elevation of CWIN activity sustained Suc import into fruits and increased activities of hexokinase and fructokinase in the ovaries in response to LMHS. Compared to the wild type, the CWIN-elevated transgenic plants exhibited higher transcript levels of heat shock protein genes Hsp90 and Hsp100 in ovaries and HspII17.6 in fruits under LMHS, which corresponded to a lower transcript level of a negative auxin responsive factor IAA9 but a higher expression of the auxin biosynthesis gene ToFZY6 in fruits at 2 d after pollination. Collectively, the data indicate that CWIN enhances fruit set under LMHS through suppression of programmed cell death in a reactive oxygen species-independent manner that could involve enhanced Suc import and catabolism, HSP expression, and auxin response and biosynthesis. Suc metabolism plays important roles in fruit and seed development through providing not only energy to power numerous cellular processes, but also sub- strates for synthesis of biopolymers such as starch and cellulose (Ruan, 2012). In parallel, Suc and hexoses derived from Suc degradation also act as signaling molecules to regulate gene expression in response to developmental and environmental cues (Wang et al., 2014; Wang and Ruan 2016). Physiologically, Suc deg- radation or utilization facilitates Suc translocation through the phloem from leaves to sinks such as developing fruits and seeds by lowering Suc concen- tration in the recipient sink cells (Ho, 1988; Ruan et al., 1995). There are two enzymes in plants to degrade Suc: Suc synthase (Sus) and invertase (INV). Sus is a glycosyl transferase and reversibly converts Suc in the presence of UDP into UDP-Glc and Fru. By contrast, INV is a hydrolase that irreversibly hydrolyses Suc into Glc and Fru. Based on its subcellular location, INV is classified into cell wall invertase (CWIN), vacuolar invertase (VIN), and cytoplasmic invertase (CIN; Sturm, 1999). CWIN activity is essential for fruit and seed devel- opment and hence crop yield (Ruan, 2014). For exam- ple, mutation of the CWIN gene INCW2 in maize (Zea mays) reduced maize grain weight by 80% (Lowe and Nelson, 1946; Miller and Chourey, 1992). Silencing CWIN expression in tomato (Solanum lycopersicum) resulted in fruit and seed abortion (Jin et al., 2009; Zanor et al., 2009). Conversely, overexpression of CWIN in rice (Oryza sativa) driven by its native promoter enhanced grain filling and yield (Wang et al., 2008). An increase in CWIN transcription or activity is also com- monly observed upon pathogen infection in a wide range of plant species (Benhamou et al., 1991; Scharte et al., 2005), indicating a role for CWIN in defense against pathogens, possibly through Glc signaling (Ruan, 2014). For instance, constitutive expression of a yeast (Saccha- romyces cerevisiae) INV gene in the apoplasm increased tobacco (Nicotiana tabacum) resistance to potato virus Y 1 This work was supported by the Australian Research Council (DP110104931, DP120104148), The University of Newcastle (G1201299), and the National Science Foundation of China (31272156). 2 Present address: College of Horticulture and Landscape Archi- tecture, Hainan University, Haikou 570228, Hainan, China. * Address correspondence to yong-ling.ruan@newcastle.edu.au. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the Journal policy described in the Instructions for Authors (www.plantphysiol.org) is: Yong-Ling Ruan (yong-ling.ruan@newcastle.edu.au). Y.-L.R. conceived the project; Y.-L.R. and C.E.O. supervised the work; Y.-H.L. performed the experiments; Y.-H.L., C.E.O., and Y.-L.R. analyzed the data; Y.-H.L. and Y.-L.R. wrote the manuscript; Y.-H.L., C.E.O., Y.-L.R. revised the manuscript. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.16.00959 Plant Physiology Ò , September 2016, Vol. 172, pp. 163–180, www.plantphysiol.org Ó 2016 American Society of Plant Biologists. All rights reserved. 163