Intracellular Na⁺/H⁺ (NHX) antiporters have important roles in cellular pH and Na⁺, K⁺ homeostasis. The six Arabidopsis thaliana intracellular NHX members are divided into two groups, endosomal (NHX5 ...and NHX6) and vacuolar (NHX1 to NHX4).Of the vacuolar members, NHX1 has been characterized functionally, but the remaining members have largely unknown roles. Using reverse genetics, we show that, unlike the single knockouts nhx1 or nhx2, the double knockout nhx1 nhx2 had significantly reduced growth, smaller cells, shorter hypocotyls in etiolated seedlings and abnormal stamens in mature flowers. Filaments of nhx1 nhx2 did not elongate and lacked the ability to dehisce and release pollen, resulting in a near lack of silique formation. Pollen viability and germination was not affected. Quantification of vacuolar pH and intravacuolar K⁺ concentrations indicated that nhx1 nhx2 vacuoles were more acidic and accumulated only 30% of the wild-type K⁺ concentration, highlighting the roles of NHX1 and NHX2 in mediating vacuolar K⁺/H⁺ exchange. Growth under added Na⁺,but not K⁺,partly rescued the flower and growth phenotypes. Our results demonstrate the roles of NHX1 and NHX2 in regulating intravacuolar K⁺ and pH, which are essential to cell expansion and flower development.
•Chitosan/alginate based LBL edible coating extended shelf life of ‘Kosui’ pear fruit.•The LBL edible coating suppressed fruit ethylene production and respiration rates.•The LBL edible coating did ...not affect fruit weight loss.•Genes related with ethylene production, softening, and color change were repressed.•Glycolytic pathway genes were upregulated, while TCA cycle genes were repressed.
Edible coatings attract much interest today as safe and effective techniques for maintaining quality and extending shelf life of fruit and vegetables. However, the underlying molecular mechanisms remain unclear. The aim of this study was to identify genes involved in enhancement of shelf life in ‘Kosui’ Japanese pear (Pyrus pyrifolia Nakai) fruit by chitosan/alginate-based layer-by-layer (LBL) edible coatings. Both tri-layer and penta-layer LBL coatings effectively minimized fruit respiration and ethylene production rates, inhibited flesh firmness loss, and prevented peel color change for 21 d at 20 °C but had little effect on weight loss. Gas permeability tests on polyethylene terephthalate films revealed that LBL coatings substantially lowered oxygen transmission rates but had little effect on water vapor transmission rate. In an attempt to identify the pathways involved, we monitored gene expression in LBL-coated and uncoated fruit by RNA-Seq analysis. This analysis revealed a clear downregulation of genes associated with ethylene production and fruit ripening, as well as the tricarboxylic acid cycle following application of LBL edible coating. On the other hand, genes associated with the glycolysis pathway were mostly upregulated. Together, these results indicate that prolonged shelf life by chitosan/alginate-based LBL edible coating could primarily result from control of the balance of aerobic-anaerobic metabolism.
In the evolutionary history of plants, variation in cis-regulatory elements (CREs) resulting in diversification of gene expression has played a central role in driving the evolution of ...lineage-specific traits. However, it is difficult to predict expression behaviors from CRE patterns to properly harness them, mainly because the biological processes are complex. In this study, we used cistrome datasets and explainable convolutional neural network (CNN) frameworks to predict genome-wide expression patterns in tomato (Solanum lycopersicum) fruit from the DNA sequences in gene regulatory regions. By fixing the effects of trans-acting factors using single cell-type spatiotemporal transcriptome data for the response variables, we developed a prediction model for crucial expression patterns in the initiation of tomato fruit ripening. Feature visualization of the CNNs identified nucleotide residues critical to the objective expression pattern in each gene, and their effects were validated experimentally in ripening tomato fruit. This cis-decoding framework will not only contribute to the understanding of the regulatory networks derived from CREs and transcription factor interactions, but also provides a flexible means of designing alleles for optimized expression.
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 ºC, moderately low storage temperatures of 5-20 °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.
Fruit quality is determined by multiple complex traits, which are difficult to diagnose by simple criteria and often require expert skills with a long experience. Nevertheless, current fruit sorting ...systems need a non-destructive, costless, and more rapid evaluation of fruit qualities. For peach, although many techniques have been developed for diagnosing fruit (internal) traits that determine commercial values, those techniques often require special facilities with high costs or take a long time for an assessment. Our study aimed to apply deep learning technology to evaluate multiple peach fruit traits using only simple RGB images for practical applications. We targeted seven fruit traits fundamentally involving commercial fruit quality; skin color, flesh firmness, sugar content, and four internal disorders, including colorless early softening, split-pit, watercore, and damage from peach fruit moth. We performed binary classification and regression analysis for these traits by convolutional neural networks (CNNs). Binary classification is performed to judge whether a fruit trait exceeds a threshold or not for a given image. Regression analysis is performed to estimate the degree of a trait quantitatively. Their results suggested that CNNs can successfully diagnose multiple fruit traits and predict quantitative values from RGB images. We also applied an explainable AI (X-AI) technique to spot the hypothetical symptoms for each trait on a fruit image, giving novel interpretations for physiological reactions associated with each fruit trait.
•Implementable peach fruit sorting needs automatic evaluation for multiple traits.•With CNN models, seven important fruit traits were diagnosable from photo images.•Xception regression model allowed estimation of quantitative traits in peach fruit.•Explainable AI technique could spot the premonitory symptoms of fruit disorders.
A major challenge in terms of commercializing 1-methylclopropene (1-MCP) to extend the storage life and control physiological disorders in European pears is that it irreversibly inhibits fruit ...ripening in some cultivars, particularly flesh softening that is necessary for optimal consumption quality. In this study, we examined the effect of 1-MCP pretreatments on fruit ripening and associated transcriptomic changes in ‘La France’ (Pyrus communis L.) pears during storage at 20°C and 5°C. Compared to non-treated controls, 1-MCP pretreatment suppressed fruit respiration and ethylene production rates, and markedly delayed flesh softening. Normal ripening (ethylene production and flesh softening to eating quality firmness) was observed in 1-MCP treated fruit after 42 d at 20°C, and 112 d at 5°C. Subsequent RNA-Seq analysis revealed that 6,427 genes, including those associated with ethylene biosynthesis (ACS1, ACS1b, ACO1, and ACO2), cell wall degrading enzymes (PG3, β-GAL, EG, and EXP1), and transcription factors (AGL18 and NAC29) were up- or down-regulated in non-treated fruit both at 20°C and 5°C. The expression patterns of these genes were disrupted by 1-MCP pretreatment, but up- or down-regulation was also observed when ethylene was detected in 1-MCP-treated fruit. Together, these findings demonstrate the potential for practical use of 1-MCP to extend storage life in ‘La France’ pears given that (i) a single application markedly extended storage life to 56 d at 20°C and 112 d at 5°C, and (ii) treated fruit could regain their softening capacity, thus eliminating previous irreversible ripening blockage concerns.
In contrast to the progress in the research on physiological disorders relating to shelf life in fruit crops, it has been difficult to non-destructively predict their occurrence. Recent high-tech ...instruments have gradually enabled non-destructive predictions for various disorders in some crops, while there are still issues in terms of efficiency and costs. Here, we propose application of a deep neural network (or simply deep learning) to simple RGB images to predict a severe fruit disorder in persimmon, rapid over-softening. With 1,080 RGB images of ‘Soshu’ persimmon fruits, three convolutional neural networks (CNN) were examined to predict rapid over-softened fruits with a binary classification and the date to fruit softening. All of the examined CNN models worked successfully for binary classification of the rapid over-softened fruits and the controls with > 80% accuracy using multiple criteria. Furthermore, the prediction values (or confidence) in the binary classification were correlated to the date to fruit softening. Although the features for classification by deep learning have been thought to be in a black box by conventional standards, recent feature visualization methods (or “explainable” deep learning) has allowed identification of the relevant regions in the original images. We applied Grad-CAM, Guided backpropagation, and layer-wise relevance propagation (LRP), to find early symptoms for CNNs classification of rapid over-softened fruits. The focus on the relevant regions tended to be on color unevenness on the surface of the fruit, especially in the peripheral regions. These results suggest that deep learning frameworks could potentially provide new insights into early physiological symptoms of which researchers are unaware.
•Kiwifruit ripening was modulated by low temperature independent of ethylene.•Fruit ripening and gene expression were induced in ‘Sanuki Gold’ fruit at ≤15 °C.•Fruit ripening and gene expression were ...induced in ‘Hayward’ fruit at ≤10 °C.•Unlike ‘Hayward’, ‘Sanuki Gold’ fruit were highly sensitive to low temperature.•Low temperature sensitivity was related to storability and on–vine maturity dates.
Kiwifruit exhibit a peculiar ripening pattern, as extensive softening is known to occur in the absence of any detectable ethylene. We previously demonstrated that this softening is regulated by low temperature independent of ethylene. However, there are no reports that provide comparisons of the ripening patterns among different kiwifruit cultivars at various storage temperatures. The purpose of this study was to compare the ripening responses and associated gene expression in ‘Sanuki Gold’ (Actinidia chinensis var. chinensis) and ‘Hayward’ ((Actinidia chinensis var. deliciosa) fruit, two kiwifruit cultivars differing in on–vine maturity dates and postharvest storability, during storage at 5 °C, 10 °C, 15 °C and 22 °C. Fruit softening, soluble solids concentration (SSC) increase and reduction of titratable acidity (TA) occurred in the absence of any detectable ethylene, and treatment with an ethylene inhibitor 1–methylcyclopropene (1–MCP) failed to suppress the changes, suggesting that they were independent of ethylene. ‘Sanuki Gold’ fruit showed a higher sensitivity to low temperature supported by accelerated fruit softening and TA reduction, and induction of several genes such as AcACO3, AcXET2, AcPG, AcEXP1, AcPMEi, AcGA2ox1, AcMADS2, AcNAC5 and AcbZIP2 at 5 °C, 10 °C and 15 °C within 28 d. By contrast, ‘Hayward’ fruit exhibited a lower sensitivity to low temperature as accelerated softening, TA reduction and induction of most ripening–associated genes were recorded only at 5 °C and 10 °C. These differences in sensitivity to low temperature, between ‘Sanuki Gold’ and ‘Hayward’ fruit, would account for the dissimilarities observed in on–vine maturity dates and postharvest storability.
Fruit ripening in response to propylene (an ethylene analog), 1-methylcyclopropene (1-MCP, an ethylene action inhibitor), and low temperature (5°C) treatments was characterized in "Kosui" kiwifruit (
...×
). Propylene treatment induced ethylene production, with increased expression levels of
(
)
(
) and
(
), and rapid fruit softening together with increased expression levels of
(
) and
(
) within 5 days (d). Fruit soluble solids concentration (SSC) and contents of sucrose, glucose, and fructose together with the expression levels of
(
),
, and
(
) increased rapidly after 5 d exposure to propylene. Furthermore, propylene exposure for 5 d was sufficient to induce the production of key aroma volatile compounds, ethyl- and methyl butanoate, accompanied with increased expression levels of
(
). Application of 1-MCP at the start of the experiment, followed by continuous exposure to propylene, significantly delayed fruit softening, changes in SSC and sugars, and strongly suppressed the production of ethylene, aroma volatiles, and expression of associated genes. During storage, fruit softening, SSC and sugar increase, and increased expression of genes associated with cell wall modification and carbohydrate metabolism were registered without detectable ethylene production; however, these changes occurred faster at 5°C compared to 22°C. Interestingly, ethyl and methyl butanoate as well as
expression were undetectable in kiwifruit during storage, while they were rescued by post-storage propylene exposure, indicating that the production of aroma volatile compounds is strongly ethylene-dependent. Transcript levels of a NAC-related transcription factor (TF),
, increased in response to both propylene and low temperature treatments, while
was exclusively up-regulated by propylene. By contrast, transcript levels of a MADS-box TF,
, exclusively increased in response to low temperature. The above findings indicate that kiwifruit ripening is inducible by either ethylene or low temperature signals. However, fruit ripened by low temperature were deficient in ethylene-dependent aroma volatiles, suggesting that ethylene signaling is non-functional during low temperature-modulated ripening in kiwifruit. These data provide further evidence that ethylene-dependent and low temperature-modulated ripening in kiwifruit involve different regulatory mechanisms.
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