Examining the Role of Low Temperature in Satsuma Mandarin Fruit Peel Degreening via Comparative Physiological and Transcriptomic Analysis

Peel degreening is the most conspicuous aspect of fruit ripening in many citrus fruits because of its importance for marketability. In this study, peel degreening in response to propylene (an ethylene analog) and at varying storage temperatures was characterized in Satsuma mandarin (Citrus unshiu Marc.) fruit. Propylene treatment triggered rapid peel degreening (within 4-6 days), indicated by an increase in the citrus color index (CCI) and chlorophyll loss. Peel degreening was also observed in fruit at 10 degrees C and 15 degrees C after 28-42 days, with gradual CCI increase and chlorophyll reduction. However, fruit at 5 degrees C, 20 degrees C, and 25 degrees C remained green, and no substantial changes in peel CCI and chlorophyll content were recorded during the 42-day storage duration. The transcriptomes of peels of fruit treated with propylene for 4 days and those stored at varying temperatures for 28 days were then analyzed by RNA-Seq. We identified three categories of differentially expressed genes that were regulated by (i) propylene (and by analogy, ethylene) alone, (ii) low temperature (5 degrees C, 10 degrees C, or 15 degrees C vs. 25 degrees C) alone, and (iii) either propylene or low temperature. Gene-encoding proteins associated with chlorophyll degradation (such as CuSGR1, CuNOL, CuACD2, CuCAB2, and CuLHCB2) and a transcription factor (CuERF114) were differentially expressed by propylene or low temperature. To further examine temperature-induced pathways, we also monitored gene expression during on-tree fruit maturation vs. postharvest. The onset of on-tree peel degreening coincided with autumnal drops in field temperatures, and it was accompanied by differential expression of low temperature-regulated genes. On the contrary, genes that were exclusively regulated by propylene (such as CuCOPT1 and CuPOX-A2) displayed insignificant expression changes during on-tree peel degreening. These findings indicate that low temperatures could be involved in the fruit ripening-related peel degreening independently of ethylene

Pollination, pollen tube growth, and fertilization independently contribute to fruit set and development in tomato

In flowering plants, pollination, pollen tube growth, and fertilization are regarded as the first hierarchical processes of producing offspring. However, their independent contributions to fruit set and development remain unclear. In this study, we examined the effect of three different types of pollen, intact pollen (IP), soft X-ray-treated pollen (XP) and dead pollen (DP), on pollen tube growth, fruit development and gene expression in “Micro-Tom” tomato. Normal germination and pollen tube growth were observed in flowers pollinated with IP; pollen tubes started to penetrate the ovary at 9 h after pollination, and full penetration was achieved after 24 h (IP24h), resulting in ~94% fruit set. At earlier time points (3 and 6 h after pollination; IP3h and IP6h, respectively), pollen tubes were still in the style, and no fruit set was observed. Flowers pollinated with XP followed by style removal after 24 h (XP24h) also demonstrated regular pollen tubes and produced parthenocarpic fruits with ~78% fruit set. As expected, DP could not germinate and failed to activate fruit formation. Histological analysis of the ovary at 2 days after anthesis (DAA) revealed that IP and XP comparably increased cell layers and cell size; however, mature fruits derived from XP were significantly smaller than those derived from IP. Furthermore, there was a high correlation between seed number and fruit size in fruit derived from IP, illustrating the crucial role of fertilization in the latter stages of fruit development. RNA-Seq analysis was carried out in ovaries derived from IP6h, IP24h, XP24h and DP24h in comparison with emasculated and unpollinated ovaries (E) at 2 DAA. The results revealed that 65 genes were differentially expressed (DE) in IP6h ovaries; these genes were closely associated with cell cycle dormancy release pathways. Conversely, 5062 and 4383 DE genes were obtained in IP24h and XP24h ovaries, respectively; top enriched terms were mostly associated with cell division and expansion in addition to the ‘plant hormone signal transduction’ pathway. These findings indicate that full penetration of pollen tubes can initiate fruit set and development independently of fertilization, most likely by activating the expression of genes regulating cell division and expansion

Low temperature modulates natural peel degreening in lemon fruit independently of endogenous ethylene

Peel degreening is an important aspect of fruit ripening in many citrus fruit, and previous studies have shown that it can be advanced by ethylene treatment or by low-temperature storage. However, the important regulators and pathways involved in natural peel degreening remain largely unknown. To determine how natural peel degreening is regulated in lemon fruit (Citrus limon), we studied transcriptome and physiochemical changes in the flavedo in response to ethylene treatment and low temperatures. Treatment with ethylene induced rapid peel degreening, which was strongly inhibited by the ethylene antagonist, 1-methylcyclopropene (1-MCP). Compared with 25 degrees C, moderately low storage temperatures of 5-20 degrees C also triggered peel degreening. Surprisingly, repeated 1-MCP treatments failed to inhibit the peel degreening induced by low temperature. Transcriptome analysis revealed that low temperature and ethylene independently regulated genes associated with chlorophyll degradation, carotenoid metabolism, photosystem proteins, phytohormone biosynthesis and signalling, and transcription factors. Peel degreening of fruit on trees occurred in association with drops in ambient temperature, and it coincided with the differential expression of low temperature-regulated genes. In contrast, genes that were uniquely regulated by ethylene showed no significant expression changes during on-tree peel degreening. Based on these findings, we hypothesize that low temperature plays a prominent role in regulating natural peel degreening independently of ethylene in citrus fruit

Combined signal sequence trap and macroarray analysis identifies genes associated with differential fruit softening characteristics during ripening in European and Chinese pears

During ripening, European pear (Pyrus communis L. cv. ‘La France’) fruit undergo dramatic softening in response to increased ethylene production, whereas Chinese pear (Pyrus bretschneideri Rehd. cv. ‘Yali’) fruit remain firm, despite producing large amounts of ethylene. The molecular basis of this differential softening behavior is not well understood. In this study, we combined a yeast-based signal sequence trap (YSST) and macroarray gene expression analysis to identify putative genes encoding secreted proteins that control pear fruit softening. We identified 22 cDNAs annotated as encoding proteins with diverse cell wall-associated functions that were up- or down-regulated during fruit ripening in ‘La France’. Gene expression analysis in fruit that were treated with the ethylene perception inhibitor 1-methylcyclopropene (1-MCP) at 4 d after the onset of ripening revealed that 16 of the targeted genes are ethylene-regulated, while the others appear to be ethylene independent. Comparative gene expression analyses of ‘La France’ and ‘Yali’ fruit during ripening suggested that four ethylene-regulated cDNAs encoding cell wall modifying proteins, contig 2 (polygalacturonase 3), contig 15 (expansin), contig 19 (expansin) and contig 55 (pectate lyase) contribute to the different softening behaviors of ‘La France’ and ‘Yali’ fruit. Additionally, one ethylene-independent cell wall related gene, contig 36 (expansin), and three genes encoding proteins of unknown function, contigs 1, 13 and contig 75 showed differential expression between ‘La France’ and ‘Yali’ fruit during ripening. The results presented herein represent promising candidates for future functional analysis and elucidation of softening mechanisms