Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsistz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC–MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome data mining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain-filling period.

Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved activesite Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsis tz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC-MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome datamining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain filling period. Short title: Suicidal and catalytic plant THI4 enzymes

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Storage at the putative chilling threshold temperature (CTT) to avoid chilling injury still limits postharvest handling of tropical fruit like banana in that ripening may occur at the CTT. To determine whether chilling injury (CI) symptoms would develop in mature green (MG) banana fruit if the CTT exposure was extended by inhibiting ethylene action and thus ripening, 1-methylcyclopropene (1-MCP) was applied. Individual ‘fingers’ from multiple ‘clusters’ of MG bananas were either immersed in water or 50 μg L−1 1-MCP (a.i.) solution and each treatment was divided into three subgroups for storage at 5.0°C (severe CI), 13.0°C (mild CI), or 14.0°C (CTT) ± 0.1°C. 1-MCP delayed ripening in terms of color change for 10 days for fruit stored at the CTT. Ethylene production by fruit at 5.0°C remained around 0.04 ng kg−1 s−1 with no obvious increase during 31-day storage. Ethylene production at 14.0°C (−1-MCP/+1-MCP) increased on Day 33 while increasing on Day 38 for 13.0°C fruit without 1-MCP and on Day 39 for fruit with 1-MCP. Peak climacteric ethylene occurred on Days 44 and 39 for 13.0 and 14.0°C fruit without 1-MCP, respectively, and on Days 59 and 51 for 13.0°C and 14.0°C 1-MCP-treated fruit, respectively. As hypothesized, longer exposure of MG banana fruit to the CTT of 14.0°C without onset of ripening as was allowed by prior 1-MCP treatment allowed CI to develop at that normally non-chilling temperature. Vascular browning was the first visual and most sensitive CI symptom in the experiment and was observed on Day 4 at 5.0°C, Day 10 at 13.0°C, Day 19 at 14.0°C without 1-MCP, and on Day 28 at 14.0°C with 1-MCP. Using a 1-MCP pre-treatment to remove the influence of ethylene from bananas stored at 13°C or 14°C also resulted in slight reduction in vascular browning severity. In conclusion, a putative safe temperature may become a CI temperature if the shelf-life-limiting factor is removed, allowing longer exposure. Chilling at the CTT caused relatively mild injury on fruit, and vascular browning is a sensitive indicator of CI status, while the light-adapted quantum yield of photosystem II [Y(II)] could be a non-destructive indicator of early CI stress in MG banana. Fruit at 13.0/14.0°C developed CI symptoms slightly later with 1-MCP than without 1-MCP. This suggests that ethylene might be involved in early CI symptom development.

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Bruising of strawberry (Fragaria ×ananassa Duch.) fruit is a common mechanical injury that reduces product value. Wound-induced ethylene may accelerate deterioration or decay, affecting strawberry quality and shelf life. However, bruising susceptibility varies among strawberry cultivars. In this study, cultivars Monterey, Sweet Sensation, Radiance, and two proprietary cultivars (Cultivar A and Cultivar B) from a private breeding program were investigated to evaluate their bruising susceptibility and wound response. Bruising consisted of dropping a 28-g steel ball from 27 cm onto individual fruit; unbruised fruit were the primary control, while fruit exposed to 1 μL·L−1 ethylene were used as a check for ethylene response. All fruit were stored at 20 °C, 90% relative humidity (RH), with respiration and ethylene production measured at 2-hour intervals for 24 hours. Appearance observations were recorded daily until decay onset. Peak respiration rates of 30–40 mL CO2·kg−1·h−1 mostly occurred within 4 hours (‘Cultivar B’) to 6 hours (‘Cultivar A’ and ‘Sweet Sensation’) after bruising, except ‘Monterey’, which peaked at 60 mL CO2·kg−1·h−1 in 2 hours, and ‘Radiance’, which reached 70 mL CO2·kg−1·h−1 in 6 hours. Maximum ethylene production rates after bruising were 0.05 to 0.06 μL·kg−1·h−1 for ‘Monterey’, ‘Cultivar A’, and ‘Cultivar B’, 0.10 μL·kg−1·h−1 for ‘Sweet Sensation’, and 0.20 to 0.37 μL·kg−1·h−1 for ‘Radiance’. ‘Cultivar B’, with the lowest ethylene production, exhibited the lowest overall bruising severity, whereas ‘Radiance’, with the highest ethylene production, exhibited the most severe bruising-induced water soaking, and the other cultivars were intermediate, although ‘Monterey’ bruises were more discolored than those of the other cultivars. ‘Monterey’, ‘Radiance’, and ‘Sweet Sensation’ showed more yellowing and browning of the calyx in response to both bruising and ethylene exposure than ‘Cultivar A’ and ‘Cultivar B’. Except for ‘Cultivar B’, bruising and ethylene exposure increased decay severity.

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