Background/Objectives: CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein 9 is now widely used in agriculture and medicine. Off-target effects can lead to unexpected results that may be harmful, and these effects are a common concern in both research and therapeutic applications. Methods: In this study, using pineapple as the gene-editing material, eighteen target sequences with varying numbers of PAM (Protospacer-Adjacent Motif) sites were used to construct gRNA vectors. Fifty mutant lines were generated for each target sequence, and the off-target rates were counted. Results: Selecting sequences with multiple flanking PAM sites as editing targets resulted in a lower off-target rate compared to those with a single PAM site. Target sequences with two 5′-NGG (“N” represents any nucleobase, followed by two guanine “G”) PAM sites at the 3′ end exhibited greater specificity and a higher probability of binding with the Cas9 protein than those only with one 5′-NGG PAM site at the 3′ end. Conversely, although the target sequence with a 5′-NAG PAM site (where “N” is any nucleobase, followed by adenine “A” and guanine “G”) adjacent and upstream of an NGG PAM site had a lower off-target rate compared to sequences with only an NGG PAM site, their off-target rates were still higher than those of sequences with two adjacent 5′-NAG PAM sites. Among the target sequences of pineapple mutant lines (AcACS1, AcOT5, AcCSPE6, AcPKG11A), more deletions than insertions were found. Conclusions: We found that target sequences with multiple flanking PAM sites are more likely to bind with the Cas9 protein and induce mutations. Selecting sequences with multiple flanking PAM sites as editing targets can reduce the off-target effects of the Cas9 enzyme in pineapple. These findings provide a foundation for improving off-target prediction and engineering CRISPR-Cas9 complexes for gene editing.

A large number of pineapple (Ananas comosus) fruits are discarded in China every year due to softening. However, the underlying molecular mechanism is still unknown. AcoPG3 (GenBank accession number: XM020243935), a pineapple gene of polygalacturonase, was found to be the major gene responsible for the softening of pineapple fruit. Fruit of AcoPG3-overexpressing tomato (Solanum lycopersicum var. Jingfan 101) begins to soften 9 days earlier than that transformed with a net vector. Fruit of AcoPG3-overexpressing pineapple (APG3-2) begins to soften 6 days earlier than that transformed with a net vector. Fruit of MPG3-1, a pineapple line in which AcoPG3 is mutated, begins to soften 31 days later than that transformed with a net vector. The sequence of polygalacturonase activities in fruit from the highest to the lowest was APG3-2, wild type, MPG3-1. The same sequences were also found in the liquid content of apoplast and the electrolyte leakage of pineapple pulp. The order of methyl-esterified pectin content in the pulp cell wall, from the highest to the lowest, was MPG3-1, wild type, and APG3-2. The same order was also observed for the contents of non-methyl-esterified homogalacturonan and rhamnogalacturonan-I in the pulp cell wall. The AcoPG3 mutation resulted in a decrease in polygalacturonase activity in pineapple fruit, decreasing the degradation of methyl-esterified pectin, non-methyl-esterified homogalacturonan, galactan and rhamnogalacturonan-I in the pulp cell wall. Fruit softening can be deferred, and the shelf life can be extended by mutating the AcoPG3 gene.

Flesh translucency is a significant physiological disorder in pineapple cultivation. Calcium deficiency in the fruit is a primary cause of flesh translucency. However, simply adding calcium to the soil or applying calcium fertilizer to the leaves and fruits does not eliminate flesh translucency. The mechanism behind this phenomenon has not been previously documented. Our research discovered that the calcium content in the stalk was significantly higher than in the fruit. Calcium ions are primarily transported through the phloem. The calcium concentration in the phloem sap of pineapple stalk at different stages ranged from 14 to 17 µmol/L. The transporter responsible for unloading Ca2+ across the membrane of the end sieve vessel in the stalk phloem is the Ca2+/H+ antiporter exchanger (CAX). Among the four CAX genes in pineapple, only AcoCAX2 is expressed in the stalk. The AcoCAX2 protein is located in the plasma membrane and cytoplasm. Calcium-sensitive yeast K667 transformed with AcoCAX2 absorbed more calcium ions from the medium compared to the control. The promoter proNtPRB1b-proAcoCAX2 is specifically expressed in the junction between the stalk and fruit. Plants overexpressing AcoCAX2 in this junction absorbed more calcium in the fruit, resulting in a lower incidence of translucency compared to the control. Fruit with a mutant AcoCAX2 gene contained less calcium and exhibited a higher incidence of translucency than control. The mutant flesh with AcoCAX2 contained more liquid in the intercellular space compared to the wild type and control. AcoCAX2 is the main gene responsible for transferring calcium ions from the stalk phloem into the fruit.

Introduction: Pineapple fruits are frequently discarded because of softening. Non-climacteric fruit ripening is regulated by abscisic acid. Pineapple is a non-climacteric fruit. Ethylene participates in pineapple ripening. However, whether abscisic acid regulates pineapple ripening and the relationship between abscisic acid and ethylene in pineapple fruit have not been reported. Methods: Pineapple mutant lines were generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The ethylene content, abscisic acid content, respiration rate, fruit firmness, aroma profiles, and the activities of polygalacturonase, pectin methylesterase, β-galactosidase, xylanase, and cellulase in pineapple flesh were measured. Quantitative real-time reverse transcription polymerase chain reaction was performed to analyze the expression of the 1-aminocyclopropane-1-carboxylate synthase gene AcoACS1 across different pineapple lines. Results and discussion: Both abscisic acid and ethylene can induce pineapple ripening and softening. Ethylene content in pineapple fruits can be stimulated by abscisic acid. An abscisic-acid-responsive element in the enhancer sequence of the 1-aminocyclopropane-1-carboxylic acid synthase gene AcoACS1 was identified. After this element was knocked out, the expression of AcoACS1 was not induced by abscisic acid treatment, and the onset of pineapple softening was delayed. Aroma compound contents in the mutant fruit and Tainong 17 were similar. Abscisic acid induces pineapple ripening via the abscisic-acid-responsive element in AcoACS1. Pineapples with a long shelf-life can be generated through knocking out this element in AcoACS1.

Background: Pineapple is the third most widely cultivated tropical fruit globally and the fourth in China. Annual losses due to translucency account for approximately 20% of China’s pineapple production. Studying its mechanism and developing controlling strategies is important for the industry. Chilling during fruit enlargement often leads to severe translucency, and larger fruits tend to show higher incidence of translucency. However, the underlying mechanisms remain unclear. Results: 15℃ treatment during fruit enlargement increased apoplastic fructose and glucose levels and up-regulated expression of the cell wall invertase gene AcoINV3 . The fructose and glucose content in pulp intercellular space of AcoINV3 mutant MU-2 is significantly less than that of the pineapple transformed with the empty vector. These values of MU-2 treated with 15 ℃ were similar with those of MU-2 always grown in 25℃ greenhouse. Under 15℃-stress, MU-2 exhibited significantly lower translucency rates than empty-vector control. Its single-fruit weight was also much less than the control. A cold shock element (CSE, 5′-CCGAC-3′) in the AcoINV3 promoter was identified as a 15℃-responsive cis -element. The AcoINV3 CSE-knockout line CSE-3 exhibited a significantly lower incidence of translucency than wild-type plants when subjected to 15℃ during fruit enlargement. Its single-fruit weight was comparable to that of wild-type plants under 25℃ conditions. Conclusions: These results suggest that 15℃ treatment during fruit enlargement induces translucency and promotes fruit weight. This inducement was associated with the CSE in the AcoINV3 promoter. The CSE in the AcoINV3 promoter has 15℃-responsive function. Targeted knockout of the CSE reduces translucency incidence without compromising single-fruit weight under 15℃ condition.