Comparative physiological and transcriptomic analysis reveal MdWRKY75 associated with sucrose accumulation in postharvest 'Honeycrisp' apples with bitter pit.

BACKGROUND: Calcium (Ca) deficiency can cause apple bitter pit, reduce the quality and shelf life. WRKY transcription factors play essential role in plant response to multiple disorders. However, the underlying mechanisms causing bitter pit in apple fruit due to Ca deficiency during storage is extremely limited. RESULTS: In the present study, the nutritional metabolites and reactive oxygen species (ROS) were compared in Ca-deficient and healthy apple fruit (CK) during storage. Results showed that Ca-deficient apples sustained significantly higher production of ROS, PPO activity, flavonoids, total phenol, total soluble solids (TSS), and sucrose contents, but the contents of Ca, H2O2, titratable acids (TA), glucose and fructose were significantly lower than those of CK during storage. Principal component analysis (PCA) showed that TSS, •O2-, PPO, malondialdehyde (MDA) and Ca were the main factors, and TSS had a positive correlation with sucrose. Furthermore, transcriptome analysis revealed that WRKYs were co-expressed with sucrose metabolism-related enzymes (SWEETs, SS, SPS). qRT-PCR and correlation analysis indicated that MdWRKY75 was correlated positively with MdSWEET1. Moreover, transient overexpression of MdWRKY75 could significantly increase the sucrose content and promote the expression of MdSWEET1 in apple fruit. CONCLUSIONS: Calcium deficiency could decrease antioxidant capacity, accelerate nutritional metabolism and up-regulate the expression of WRKYs in apple with bitter pit. Overexpression of MdWRKY75 significantly increased sucrose accumulation and the expression of MdSWEET1. These findings further strengthened knowledge of the basic molecular mechanisms in calcium deficiency apple flesh and contributed to improving the nutritional quality of apple fruit.

Dissection and ultramicroscopic observation of an apical pollen tube of Pyrus.

The pollen tube is ideal for studying cell polar growth, and observing the ultrastructure of the pollen tube tip using transmission electron microscopy (TEM) is the primary method for studying pollen tube growth. The preparation of ultrathin sections of the pollen tube tip sample is important for its successful microscopic observation. The direction of pollen tube growth in vitro is irregular, and it is difficult to dissect the tip of the pollen tube during ultrathin sectioning. Here, we used two methods to efficiently obtain an ultrathin section of the pollen tube tip of Pyrus. In the first method, laser micro-cutting was used to obtain the pollen tube tip, followed by ultrathin sectioning. In the other method, the pollen tubes were cultured in the same growth direction, followed by ultrathin sectioning. Ultrathin sections, which were observed via TEM, showed typical characteristics of the pollen tube tip, such as dense vesicles, numerous mitochondria, and secretory vesicles of the Golgi. We concluded that these two methods are effective in pollen tube tip sample preparation for ultrathin sectioning and provide the foundation for observing the ultrastructure of pollen tube tips.

Transcriptomics Reveals the ERF2-bHLH2-CML5 Module Responses to H2S and ROS in Postharvest Calcium Deficiency Apples.

Calcium deficiency usually causes accelerated quality deterioration in postharvest fruit, whereas the underlining mechanism is still unclear. Here, we report that calcium deficiency induced the development of bitter pit on the surface of apple peels compared with the healthy appearance in control apples during postharvest storage. Physiological analysis indicates that calcium-deficient peels contained higher levels of superoxide anion (O2•-), malondialdehyde (MDA), total phenol, flavonoid contents and polyphenol oxidase (PPO) activity, and reduced calcium, H2S production, anthocyanin, soluble protein content, and peroxidase (POD) activity compared with those in calcium-sufficient peels. The principal component analysis (PCA) results show that calcium content, ROS, and H2S production were the main factors between calcium-deficient and calcium-sufficient apple peels. Transcriptome data indicated that four calmodulin-like proteins (CMLs), seven AP2/ERFs, and three bHLHs transcripts were significantly differentially expressed in calcium-deficient apple peels. RT-qPCR and correlation analyses further revealed that CML5 expression was significantly positively correlated with the expression of ERF2/17, bHLH2, and H2S production related genes. In addition, transcriptional co-activation of CML5 by ERF2 and bHLH2 was demonstrated by apple transient expression assays and dual-luciferase reporter system experiments. Therefore, these findings provide a basis for studying the molecular mechanism of postharvest quality decline in calcium-deficient apples and the potential interaction between Ca2+ and endogenous H2S.

Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp.

Cytosolic Ca2+ plays a key role in plant development. Calcium imaging is the most versatile method to detect dynamic changes in Ca2+ in the cytoplasm. In this study, we obtained viable protoplasts of pulp cells by enzymatic hydrolysis. Isolated protoplasts were incubated with the small-molecule fluorescent reagent (Fluo-4/AM) for 30 min at 37 °C. The fluorescent probes successfully stained cytosolic Ca2+ but did not accumulate in vacuoles. La3+, a Ca2+ channel blocker, decreased cytoplasmic fluorescence intensity. These results suggest that Fluo-4/AM can be used to detect changes in cytosolic Ca2+ in the fruit flesh. In summary, we present a method to effectively isolate protoplasts from flesh cells of the fruit and detect Ca2+ by loading a small-molecule calcium fluorescent reagent in the cytoplasm of pulp cells.

Accumulation of Abnormal Amyloplasts in Pulp Cells Induces Bitter Pit in Malus domestica.

Apple bitter pit primarily occurs during fruit ripening and storage; however, its formation mechanism remains unclear. Although it is considered that Ca2+ deficiency causes metabolic disorders in apples, there have been few studies on the mechanism of the bitter pit from the perspective of cell structure. At the fruit ripening stage, the fruit with a bitter pit on the tree was taken as the research material. In this study, the microscopic observation revealed numerous amyloplasts in the pulp cells of apples affected with bitter pit, but not in the healthy pulp. Furthermore, the results of fluorescence staining and transmission electron microscopy (TEM) revealed that the bitter pit pulp cells undergo programmed cell death (PCD), their nuclear chromosomes condense, and amyloplast forms autophagy. The cytoplasmic Ca2+ concentration in the healthy fruits was lowest near the peduncle, followed by that in the calyx, whereas it was highest at the equator. In contrast, the cytoplasmic Ca2+ concentration in apple fruits showing bitter pit disorder was lowest near the peduncle and highest in the calyx. Moreover, the cytosolic Ca2+ concentration in the flesh cells of apples with the bitter pit was much lower than that in the healthy apple flesh cells; however, the concentration of Ca2+ in the vacuoles of fruits with the bitter pit was higher than that in the vacuoles of healthy fruits. In summary, bitter pit pulp cells contain a large number of amyloplasts, which disrupts the distribution of Ca2+ in the pulp cells and causes PCD. These two processes lead to an imbalance in cell metabolism and induce the formation of a bitter pit.

Loading calcium fluorescent probes into protoplasts to detect calcium in the flesh tissue cells of Malus domestica.

Cytosolic Ca2+ plays a key role in signal transduction in plants. Calcium imaging is the most common approach to studying dynamic changes in the cytoplasmic Ca2+ content. Here, we used mature 'Fuji' apples (Malus pumila Mill.) to obtain viable protoplasts from flesh tissue cells by enzymatic hydrolysis; then, three small-molecule fluorescent probes (fluo-8/AM, fluo-4/AM, and rhod-2/AM) were loaded into the protoplasts. All three Ca2+ fluorescent probes successfully entered the cytoplasm but did not enter the vacuole. Both the Ca2+ chelator (EGTA) and Ca2+ channel blocker (La3+) reduced the fluorescence intensity in the cytoplasm. The calcium ionophore A23187 increased the fluorescence intensity in the cytoplasm at 5 µmol/L but decreased it at 50 µmol/L. Additionally, A23187 reversed the fluorescence intensity in the cytoplasm, which was decreased by La3+. Ionomycin is also a calcium ionophore that can increase the fluorescence intensity of calcium in the cytoplasm. These results suggest that small-molecule Ca2+ fluorescent probes can be used to detect changes in cytosolic calcium levels in the cells of fruit flesh tissue. In addition, the optimum concentration of fluo-8/AM was determined to be 5 µmol/L. This was the first time that protoplasts have been isolated from apple flesh tissue cells and small-molecule fluorescent probes have been used to detect calcium in the cytoplasm of flesh tissue cells. This study provides a new method to study calcium signal transduction in fruit flesh tissue.

RNA-Seq analysis of compatible and incompatible styles of Pyrus species at the beginning of pollination.

KEY MESSAGE: At the early stage of pollination, the difference in gene expression between compatibility and incompatibility is highly significant about the pollen-specific expression of the LRR gene, resistance, and defensin genes. In Rosaceae, incompatible pollen can penetrate into the style during the gametophytic self-incompatibility response. It is therefore considered a stylar event rather than a stigmatic event. In this study, we explored the differences in gene expression between compatibility and incompatibility in the early stage of pollination. The self-compatible pear variety "Jinzhuili" is a naturally occurring bud mutant from "Yali", a leading Chinese native cultivar exhibiting typical gametophytic self-incompatibility. We collected the styles of 'Yali' and 'Jinzhuili' at 0.5 and 2 h after self-pollination and then performed high-throughput sequencing. According to the KEGG analysis of the differentially expressed genes, several metabolic pathways, such as "Plant hormone signal transduction", "Plant-pathogen interaction", are the main pathways was the most represented pathway. Quantitative PCR was used to validate these differential genes. The expression levels of genes related to pollen growth and disease inhibition, such as LRR (Leucine-rich repeat extensin), resistance, defensin, and auxin, differed significantly between compatible and incompatible pollination. Interestingly, at 0.5 h, most of these genes were upregulated in the compatible pollination system compared with the incompatible pollination system. Calcium transport, which requires ATPase, also demonstrated upregulated expression. In summary, the self-incompatibility reaction was initiated when the pollen land on the stigma.

The changes of intracellular calcium concentration and distribution in the hard end pear (Pyrus pyrifolia cv. 'Whangkeumbae') fruit.

Hard end is a physiological disorder of pear fruit that is frequently observed in the 'Whangkeumbae' (Pyrus pyrifolia) variety, however, the mechanisms that are involved in its development are poorly understood. In this study, we explored the causes of hard end disorder in pear fruit in relation to calcium deficiency. During fruit development, the ratio of Ca/N, Ca/K, Ca/Mg and the content of B were significantly lower in the hard end fruit as compared to normal fruit. However, no calcium deficiency was detected in the soil and leaves of the orchard where the hard end fruit were located. Additionally, the Ca2+ influx in the calyx of hard end fruit was lower than that of normal fruit at 90 d after anthesis. The free Ca2+ and storage Ca2+ in the flesh cells of hard end fruit were less than that of normal fruit during fruit development, while an opposite tendency was observed at 120 d after anthesis (harvest day). In hard end fruit, the Ca2+ transport-related gene, PpCNGC1 (Cyclic nucleotide-gated ion channel 1), was up-regulated; whereas the Ca2+ sensor-related genes of PpCIPKs, PpCDPK28 and PpCML41 were all down-regulated. Spraying with a 2% calcium chloride (CaCl2) solution inhibited the incidence rate of hard end disorder and decreased fruit firmness and lignin content during storage. Additionally, the ratio of Ca/N, Ca/K, Ca/Mg and the content of B all increased on harvest day. Our study suggests that low Ca2+ influx leads to less Ca2+ into the pear fruit, which results in an intracellular imbalance of Ca2+ and consequently triggers the development of hard end disorder.

PLC-Mediated Signaling Pathway in Pollen Tubes Regulates the Gametophytic Self-incompatibility of Pyrus Species.

Among the Rosaceae species, the gametophytic self-incompatibility (GSI) is controlled by a single multi-allelic S locus, which is composed of the pistil-S and pollen-S genes. The pistil-S gene encodes a polymorphic ribonuclease (S-RNase), which is essential for identifying self-pollen. However, the S-RNase system has not been fully characterized. In this study, the self-S-RNase inhibited the Ca2+-permeable channel activity at pollen tube apices and the selectively decreased phospholipase C (PLC) activity in the plasma membrane of Pyrus pyrifolia pollen tubes. Self-S-RNase decreased the Ca2+ influx through a PLC-mediated signaling pathway. Phosphatidylinositol-specific PLC has a 26-amino acid insertion in pollen tubes of the 'Jinzhuili' cultivar, which is a spontaneous self-compatible mutant of the 'Yali' cultivar. 'Yali' plants exhibit a typical S-RNase-based GSI. Upon self-pollination, PLC gene expression is significantly higher in 'Jinzhuili' pollen tubes than that in 'Yali' pollen tubes. Moreover, the PLC in pollen tubes can only interact with one of the two types of S-RNase from the style. In the Pyrus x bretschneideri Rehd, the PLC directly interacted with the S7-RNase in the pollen tube, but not with the S34-RNase. Collectively, our results reveal that the effects of S-RNase on PLC activity are required for S-specific pollen rejection, and that PLC-IP3 participates in the self-incompatibility reaction of Pyrus species.

The role of Ca2+ and Ca2+ channels in the gametophytic self-incompatibility of Pyrus pyrifolia.

In S-RNase-based self-incompatibility, S-RNase was previously thought to function as a selective RNase that inhibits pollen whose S-haplotype matches that in the pistil. In this study, we showed that S-RNase has a distinct effect on the regulation of Ca2+-permeable channel activity in the apical pollen tube in Pyrus pyrifolia. While non-self S-RNase has no effect, self S-RNase decreases the activity of Ca2+ channels and disrupts the Ca2+ gradient at the tip of the growing pollen tube during the gametophytic self-incompatibility (GSI) response. Extracellular Ca2+ influx was suppressed 5min after self S-RNase treatment, and self-pollen tube growth was reduced at 50min after self S-RNase treatment. In the self-incompatible response, the expression of Ca2+-related genes was inhibited before RNA degradation. Therefore, self S-RNase suppresses Ca2+ influx prior to arresting pollen tube growth via RNA degradation.

Rapid and Inexpensive Method of Loading Fluorescent Dye into Pollen Tubes and Root Hairs.

The most direct technique for studying calcium, which is an essential element for pollen tube growth, is Ca2+ imaging. Because membranes are relatively impermeable, the loading of fluorescent Ca2+ probes into plant cells is a challenging task. Thus, we have developed a new method of loading fluo-4 acetoxymethyl ester into cells that uses a cell lysis solution to improve the introduction of this fluorescent dye into pollen tubes. Using this method, the loading times were reduced to 15 min. Furthermore, loading did not have to be performed at low (4°C) temperatures and was successful at room temperature, and pluronic F-127 was not required, which would theoretically allow for the loading of an unlimited number of cells. Moreover, the method can also be used to fluorescently stain root hairs.

Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia.

Loading of Ca(2+)-sensitive fluorescent probes into plant cells is an essential step to measure activities of free Ca(2+) ions in cytoplasm with a fluorescent imaging technique. Fluo-3 is one of the most suitable Ca(2+) indicators for CLSM. We loaded pollen with fluo-3/AM at three different temperatures. Fluo-3/AM was successfully loaded into pollen at both low (4°C) and high (37°C) temperatures. However, high loading temperature was best suited for pollen, because germination rate of pollen and growth of pollen tubes were relatively little impaired and loading time was shortened. Moreover, Ca(2+) distribution increased in the three apertures of pollen after hydration and showed a Ca(2+) gradient, similar to the tip of growing pollen tubes. The same protocol can be used with the AM-forms of other fluorescent dyes for effective labeling. When loading BCECF-AM into pollen at high temperature, the pollen did not show a pH gradient after hydration. Ca(2+) activities and fluxes had the same periodicity as pollen germination, but pH did not show the same phase and mostly lagged behind. However, the clear zone was alkaline when pollen tube growth was slowed or stopped and turned acidic when growth recovered. It is likely that apical pH(i) regulated pollen tube growth.

cAMP activates hyperpolarization-activated Ca2+ channels in the pollen of Pyrus pyrifolia.

Many signal-transduction processes in plant cells have been suggested to be triggered by signal-induced opening of calcium ion (Ca(2+)) channels in the plasma membrane. Cyclic nucleotides have been proposed to lead to an increase in cytosolic free Ca(2+) in pollen. However, direct recordings of cyclic-nucleotide-induced Ca(2+) currents in pollen have not yet been obtained. Here, we report that cyclic AMP (cAMP) activated a hyperpolarization-activated Ca(2+) channel in the Pyrus pyrifolia pollen tube using the patch-clamp technique, which resulted in a significant increase in pollen tube protoplast cytosolic-Ca(2+) concentration. Outside-out single channel configuration identified that cAMP directly increased the Ca(2+) channel open-probability without affecting channel conductance. cAMP-induced currents were composed of both Ca(2+) and K(+). However, cGMP failed to mimic the cAMP effect. Higher cytosolic free-Ca(2+) concentration significantly decreased the cAMP-induced currents. These results provide direct evidence for cAMP activation of hyperpolarization-activated Ca(2+) channels in the plasma membrane of pollen tubes, which, in turn, modulate cellular responses in regulation of pollen tube growth.

Reciprocal regulation of Ca²+-activated outward K+ channels of Pyrus pyrifolia pollen by heme and carbon monoxide.

• The regulation of plant potassium (K+) channels has been extensively studied in various systems. However, the mechanism of their regulation in the pollen tube is unclear. • In this study, the effects of heme and carbon monoxide (CO) on the outward K+ (K+(out)) channel in pear (Pyrus pyrifolia) pollen tube protoplasts were characterized using a patch-clamp technique. • Heme (1 µM) decreased the probability of K+(out) channel opening without affecting the unitary conductance, but this inhibition disappeared when heme was co-applied with 10 µM intracellular free Ca²+. Conversely, exposure to heme in the presence of NADPH increased channel activity. However, with tin protoporphyrin IX treatment, which inhibits hemeoxygenase activity, the inhibition of the K+(out) channel by heme occurred even in the presence of NADPH. CO, a product of heme catabolism by hemeoxygenase, activates the K+(out) channel in pollen tube protoplasts in a dose-dependent manner. The current induced by CO was inhibited by the K+ channel inhibitor tetraethylammonium. • These data indicate a role of heme and CO in reciprocal regulation of the K+(out) channel in pear pollen tubes.

Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia.

The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca(2+)](cyt)) is essential for pollen-tube growth. Local Ca(2+) influx mediated by Ca(2+)-permeable channels plays a key role in maintaining this [Ca(2+)](cyt) gradient. Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique. We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba(2+)) approximately Ca(2+) > magnesium (Mg(2+)) > strontium (Sr(2+)) > manganese (Mn(2+)). This channel conductance was selective for Ca(2+) over chlorine (Cl(-)) (relative permeability P(Ca)/P(Cl) = 14 in 10 mm extracellular Ca(2+)). We also showed that the channel was inhibited by the Ca(2+) channel blockers lanthanum (La(3+)) and gadolinium (Gd(3+)). Furthermore, channel activity depended on extracellular pH and pollen viability. We propose that the Ca(2+)-permeable channel is likely to play a role in mediating Ca(2+) influx into the growing pollen tubes to maintain the [Ca(2+)](cyt) gradient.

[Effects of nitrogen and calcium on polyphenol oxidase, peroxidase and leaf spot in carnation].

Carnation inoculated with leaf spot was cultivated under different concentration of nitrogen and calcium in this experiment. The result showed that the nutrition state [NO3- (0.024%-0.025%), Ca2+ (0.022%-0.0342%)] was helpful to keep higher activity of POD and PPO and enhance the carnation's resistance to the germs.