Emerging Roles of Mitogen-Activated Protein Kinase Signaling Pathways in the Regulation of Fruit Ripening and Postharvest Quality.

Fleshy fruit ripening is a unique biological process that involves dramatic changes in a diverse array of cellular metabolisms. The regulation of these metabolisms is essentially mediated by cellular signal transduction of internal (e.g., hormones) and external cues (i.e., environmental stimuli). Mitogen-activated protein kinase (MAPK) signaling pathways play crucial roles in a diverse array of biological processes, such as plant growth, development and biotic/abiotic responses. Accumulating evidence suggests that MAPK signaling pathways are also implicated in fruit ripening and quality formation. However, while MAPK signaling has been extensively reviewed in Arabidopsis and some crop plants, the comprehensive picture of how MAPK signaling regulates fruit ripening and quality formation remains unclear. In this review, we summarize and discuss research in this area. We first summarize recent studies on the expression patterns of related kinase members in relation to fruit development and ripening and then summarize and discuss the crucial evidence of the involvement of MAPK signaling in fruit ripening and quality formation. Finally, we propose several perspectives, highlighting the research matters and questions that should be afforded particular attention in future studies.

A signaling cascade mediating fruit trait development via phosphorylation-modulated nuclear accumulation of JAZ repressor.

It is generally accepted that jasmonate-ZIM domain (JAZ) repressors act to mediate jasmonate (JA) signaling via CORONATINE-INSENSITIVE1 (COI1)-mediated degradation. Here, we report a cryptic signaling cascade where a JAZ repressor, FvJAZ12, mediates multiple signaling inputs via phosphorylation-modulated subcellular translocation rather than the COI1-mediated degradation mechanism in strawberry (Fragaria vesca). FvJAZ12 acts to regulate flavor metabolism and defense response, and was found to be the target of FvMPK6, a mitogen-activated protein kinase that is capable of responding to multiple signal stimuli. FvMPK6 phosphorylates FvJAZ12 at the amino acid residues S179 and T183 adjacent to the PY residues, thereby attenuating its nuclear accumulation and relieving its repression for FvMYC2, which acts to control the expression of lipoxygenase 3 (FvLOX3), an important gene involved in JA biosynthesis and a diverse array of cellular metabolisms. Our data reveal a previously unreported mechanism for JA signaling and decipher a signaling cascade that links multiple signaling inputs with fruit trait development.

FvWRKY48 binds to the pectate lyase FvPLA promoter to control fruit softening in Fragaria vesca.

The regulatory mechanisms that link WRKY gene expression to fruit ripening are largely unknown. Using transgenic approaches, we showed that a WRKY gene from wild strawberry (Fragaria vesca), FvWRKY48, may be involved in fruit softening and ripening. We showed that FvWRKY48 is localized to the nucleus and that degradation of the pectin cell wall polymer homogalacturonan, which is present in the middle lamella and tricellular junction zones of the fruit, was greater in FvWRKY48-OE (overexpressing) fruits than in empty vector (EV)-transformed fruits and less substantial in FvWRKY48-RNAi (RNA interference) fruits. Transcriptomic analysis indicated that the expression of pectate lyase A (FvPLA) was significantly downregulated in the FvWRKY48-RNAi receptacle. We determined that FvWRKY48 bound to the FvPLA promoter via a W-box element through yeast one-hybrid, electrophoretic mobility shift, and chromatin immunoprecipitation quantitative polymerase chain reaction experiments, and ß-glucosidase activity assays suggested that this binding promotes pectate lyase activity. In addition, softening and pectin degradation were more intense in FvPLA-OE fruit than in EV fruit, and the middle lamella and tricellular junction zones were denser in FvPLA-RNAi fruit than in EV fruit. We speculated that FvWRKY48 maybe increase the expression of FvPLA, resulting in pectin degradation and fruit softening.

Cell Wall Integrity Signaling in Fruit Ripening.

Plant cell walls are essential structures for plant growth and development as well as plant adaptation to environmental stresses. Thus, plants have evolved signaling mechanisms to monitor the changes in the cell wall structure, triggering compensatory changes to sustain cell wall integrity (CWI). CWI signaling can be initiated in response to environmental and developmental signals. However, while environmental stress-associated CWI signaling has been extensively studied and reviewed, less attention has been paid to CWI signaling in relation to plant growth and development under normal conditions. Fleshy fruit development and ripening is a unique process in which dramatic alternations occur in cell wall architecture. Emerging evidence suggests that CWI signaling plays a pivotal role in fruit ripening. In this review, we summarize and discuss the CWI signaling in relation to fruit ripening, which will include cell wall fragment signaling, calcium signaling, and NO signaling, as well as Receptor-Like Protein Kinase (RLKs) signaling with an emphasis on the signaling of FERONIA and THESEUS, two members of RLKs that may act as potential CWI sensors in the modulation of hormonal signal origination and transduction in fruit development and ripening.

SnRK2 subfamily I protein kinases regulate ethylene biosynthesis by phosphorylating HB transcription factors to induce ACO1 expression in apple.

Ethylene (ETH) controls climacteric fruit ripening and can be triggered by osmotic stress. However, the mechanism regulating ETH biosynthesis during fruit ripening and under osmotic stress is largely unknown in apple (Malus domestica). Here, we explored the roles of SnRK2 protein kinases in ETH biosynthesis related to fruit ripening and osmoregulation. We identified the substrates of MdSnRK2-I using phosphorylation analysis techniques. Finally, we identified the MdSnRK2-I-mediated signaling pathway for ETH biosynthesis related to fruit ripening and osmoregulation. The activity of two MdSnRK2-I members, MdSnRK2.4 and MdSnRK2.9, was significantly upregulated during ripening or following mannitol treatment. Overexpression of MdSnRK2-I increased ETH biosynthesis under normal and osmotic conditions in apple fruit. MdSnRK2-I phosphorylated the transcription factors MdHB1 and MdHB2 to enhance their protein stability and transcriptional activity on MdACO1. MdSnRK2-I also interacted with MdACS1 and increased its protein stability through two phosphorylation sites. The increased MdACO1 expression and MdACS1 protein stability resulted in higher ETH production in apple fruit. In addition, heterologous expression of MdSnRK2-I or manipulation of SlSnRK2-I expression in tomato (Solanum lycopersicum) fruit altered fruit ripening and ETH biosynthesis. We established that MdSnRK2-I functions in fruit ripening and osmoregulation, and identified the MdSnRK2-I-mediated signaling pathway controlling ETH biosynthesis.

A Method for Assaying of Protein Kinase Activity In Vivo and Its Use in Studies of Signal Transduction in Strawberry Fruit Ripening.

Strawberry (Fragaria × ananassa) fruit ripening is regulated by a complex of cellular signal transduction networks, in which protein kinases are key components. Here, we report a relatively simple method for assaying protein kinase activity in vivo and specifically its application to study the kinase, FaMPK6, signaling in strawberry fruit. Green fluorescent protein (GFP)-tagged FaMPK6 was transiently expressed in strawberry fruit and after stimuli were applied to the fruit it was precipitated using an anti-GFP antibody. The precipitated kinase activity was measured in vitro using 32P-ATP and myelin basic protein (MBP) as substrates. We also report that FaMPK6 is not involved in the abscisic acid (ABA) signaling cascade, which is closely associated with FaMPK6 signaling in other plant species. However, methyl jasmonate (MeJA), low temperature, and high salt treatments were all found to activate FaMPK6. Transient manipulation of FaMPK6 expression was observed to cause significant changes in the expression patterns of 2749 genes, of which 264 were associated with MeJA signaling. The data also suggest a role for FaMPK6 in modulating cell wall metabolism during fruit ripening. Taken together, the presented method is powerful and its use will contribute to a profound exploration to the signaling mechanism of strawberry fruit ripening.

FaMYB44.2, a transcriptional repressor, negatively regulates sucrose accumulation in strawberry receptacles through interplay with FaMYB10.

Sugar and acid metabolism are critical for fruit ripening and quality formation, but the underlying regulatory mechanisms are largely unknown. Here, we identified a transcriptional repressor, FaMYB44.2, that regulates sugar and acid accumulation in strawberry (Fragaria × ananassa 'Benihoppe') receptacles. We transiently expressed FaMYB44.2 in strawberry fruit and conducted metabolic and molecular analyses to explore the role of FaMYB44.2 in sugar and acid accumulation in strawberry. We found that FaMYB44.2 negatively regulates soluble sugar accumulation and malic acid content and represses the expression of numerous structural genes, including FaSPS3, a key gene in sucrose accumulation. From the white fruit stage onwards, the repressive effect of FaMYB44.2 on FaSPS3 is reversed by FaMYB10, which positively regulates anthocyanin accumulation. Our results indicate that FaMYB10 suppresses FaMYB44.2 expression; weakens the interaction between FaMYB44.2 and its co-repressor, FabHLH3; and cooperates with FabHLH3 to activate the expression of FaSPS3. The interplay between FaMYB10 and FaMYB44.2 results in sucrose accumulation in ripe strawberry fruits. In addition, the repressive effect of FaMYB44.2 on sucrose accumulation is enhanced by jasmonic acid. This study provides new insights into the regulatory mechanisms of sucrose accumulation and sheds light on the interplay between regulatory proteins during strawberry fruit ripening and quality formation.

SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE2.6, an ortholog of OPEN STOMATA1, is a negative regulator of strawberry fruit development and ripening.

Whereas the regulatory mechanisms that direct fruit ripening have been studied extensively, little is known about the signaling mechanisms underlying this process, especially for nonclimacteric fruits. In this study, we demonstrated that a SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE2, designated as FaSnRK2.6, is a negative regulator of fruit development and ripening in the nonclimacteric fruit strawberry (Fragaria × ananassa) and can also mediate temperature-modulated strawberry fruit ripening. FaSnRK2.6 was identified as an ortholog of OPEN STOMATA1. Levels of FaSnRK2.6 transcript rapidly decreased during strawberry fruit development and ripening. FaSnRK2.6 was found to be capable of physically interacting with strawberry ABSCISIC ACID INSENSITIVE1, a negative regulator in strawberry fruit ripening. RNA interference-induced silencing of FaSnRK2.6 significantly promoted fruit ripening. By contrast, overexpression of FaSnRK2.6 arrested fruit ripening. Strawberry fruit ripening is highly sensitive to temperature, with high temperatures promoting ripening and low temperatures delaying it. As the temperature increased, the level of FaSnRK2.6 expression declined. Furthermore, manipulating the level of FaSnRK2.6 expression altered the expression of a variety of temperature-responsive genes. Taken together, this study demonstrates that FaSnRK2.6 is a negative regulator of strawberry fruit development and ripening and, furthermore, that FaSnRK2.6 mediates temperature-modulated strawberry fruit ripening.

Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress.

Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5-MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases.

AtDsPTP1 acts as a negative regulator in osmotic stress signalling during Arabidopsis seed germination and seedling establishment.

Dual-specificity protein phosphatases (DsPTPs) target both tyrosine and serine/threonine residues and play roles in plant growth and development. We have characterized an Arabidopsis mutant, dsptp1, which shows a higher seed germination rate and better root elongation under osmotic stress than the wild type. By contrast, its overexpression line, DsPTP1-OE, shows inhibited seed germination and root elongation; and its complemented line, DsPTP1-Com, resembles the wild type and rescues DsPTP1-OE under osmotic stress. Expression of AtDsPTP1 is enhanced by osmotic stress in seed coats, bases of rosette leaves, and roots. Compared with the wild type, the dsptp1 mutant shows increased proline accumulation, reduced malondialdehyde (MDA) content and ion leakage, and enhanced antioxidant enzyme activity in response to osmotic stress. AtDsPTP1 regulates the transcript levels of various dehydration-responsive genes under osmotic stress. Abscisic acid (ABA) accumulation in dsptp1 under osmotic stress is reduced with reduced expression of the ABA-biosynthesis gene NCED3 and increased expression of the ABA-catabolism gene CYP707A4. AtDsPTP1 also regulates the expression of key components in the ABA-signalling pathway. In conclusion, AtDsPTP1 regulates ABA accumulation, and acts as a negative regulator in osmotic stress signalling during Arabidospsis seed germination and seedling establishment.

Budding mutation reprogrammed flavonoid biosynthesis in jujube by deploying MYB41 and bHLH93.

Budding mutations are known to cause metabolic changes in new jujube varieties; however, the mechanisms underlying these changes are still unclear. Here, we performed muti-omics analysis to decipher the detailed metabolic landscape of "Saimisu 1" (S1) and its budding mutation line "Saimisu 2" (S2) at all fruit stages. We found that the genes involved in the biosyntheses of flavonoids, phenylpropanoids, and amino acids were upregulated in S2 fruits at all stages, especially PAL and DFR, resulting in increased accumulation of related compounds in S2 mature fruits. Further co-expression regulatory network analysis showed that the transcription factors MYB41 and bHLH93 potentially regulated the expression of PAL and DFR, respectively, by directly binding to their promoters. Moreover, the overexpression of MYB41 or bHLH93 induced their expression levels to redirect the flux of the flavonoid biosynthetic pathway, eventually leading to high levels of related compounds in S2 fruits. Overall, this study revealed the metabolic variations between S1 and S2 and contributed to the understanding of the mechanisms underlying budding mutation-mediated metabolic variations in plants, eventually providing the basis for breeding excellent jujube varieties using budding mutation lines.

MKK5 regulates high light-induced gene expression of Cu/Zn superoxide dismutase 1 and 2 in Arabidopsis.

Superoxide dismutases (SODs) convert the superoxide radical to hydrogen peroxide and molecular oxygen, and play crucial roles in plant tolerance to oxidative stress. Expression of many genes encoding SODs is promoted in response to environmental stresses, but the exact mechanism of such promotion is largely unknown. Here, we report that MKK5, a mitogen-activated protein kinase kinase, mediated the high light-induced expression of genes of two copper/zinc SODs, CSD1 and CSD2, and was involved in the oxidative adaptation to high light stress. In response to high light, wild-type Arabidopsis plants showed much enhanced expression of CSD1 and CSD2 and higher enzyme activity of MKK5. In the MKK5-RNAi (RNA interference) lines, however, the induction of CSD1 and CSD2 as well as the activation of MKK5 activity were completely arrested. In contrast, overexpression of MKK5 promoted the expression of CSD1 and CSD2. MKK5-RNAi gene silencing and CSD1/2-RNAi suppression plants became much more sensitive to high light stress than wild-type plants, and the double mutant mkk5 csd1 exhibited hypersensitivity to the stress. Plants overexpressing MKK5 showed enhanced tolerance to high light stress. Our results demonstrate that MKK5 mediated a signal of the high light-induced expression of the genes CSD1 and CSD2. Manipulating MKK5 and thereby up-regulating the levels of CSD1 and CSD2 transcripts can improve plant tolerance to high light stress.

Sucrose functions as a signal involved in the regulation of strawberry fruit development and ripening.

Fleshy fruits are classically divided into climacteric and nonclimacteric types. It has long been thought that the ripening of climacteric and nonclimacteric fruits is regulated by ethylene and abscisic acid (ABA), respectively. Here, we report that sucrose functions as a signal in the ripening of strawberry (Fragaria × ananassa), a nonclimacteric fruit. Pharmacological experiments, as well as gain- and loss-of-function studies, were performed to demonstrate the critical role of sucrose in the regulation of fruit ripening. Fruit growth and development were closely correlated with a change in sucrose content. Exogenous sucrose and its nonmetabolizable analog, turanose, induced ABA accumulation in fruit and accelerated dramatically fruit ripening. A set of sucrose transporters, FaSUT1-7, was identified and characterized, among which FaSUT1 was found to be a major component responsible for sucrose accumulation during fruit development. RNA interference-induced silencing of FaSUT1 led to a decrease in both sucrose and ABA content, and arrested fruit ripening. By contrast, overexpression of FaSUT1 led to an increase in both sucrose and ABA content, and accelerated fruit ripening. In conclusion, this study demonstrates that sucrose is an important signal in the regulation of strawberry fruit ripening.

Purification and characterization of ZmRIP1, a novel reductant-inhibited protein tyrosine phosphatase from maize.

Protein tyrosine phosphatases (PTPases) have long been thought to be activated by reductants and deactivated by oxidants, owing to the presence of a crucial sulfhydryl group in their catalytic centers. In this article, we report the purification and characterization of Reductant-Inhibited PTPase1 (ZmRIP1) from maize (Zea mays) coleoptiles, and show that this PTPase has a unique mode of redox regulation and signaling. Surprisingly, ZmRIP1 was found to be deactivated by a reductant. A cysteine (Cys) residue (Cys-181) near the active center was found to regulate this unique mode of redox regulation, as mutation of Cys-181 to arginine-181 allowed ZmRIP1 to be activated by a reductant. In response to oxidant treatment, ZmRIP1 was translocated from the chloroplast to the nucleus. Expression of ZmRIP1 in Arabidopsis (Arabidopsis thaliana) plants and maize protoplasts altered the expression of genes encoding enzymes involved in antioxidant catabolism, such as At1g02950, which encodes a glutathione transferase. Thus, the novel PTPase identified in this study is predicted to function in redox signaling in maize.

Induction of root Fe(lll) reductase activity and proton extrusion by iron deficiency is mediated by auxin-based systemic signalling in Malus xiaojinensis.

Iron is a critical cofactor for a number of metalloenzymes involved in respiration and photosynthesis, but plants often suffer from iron deficiency due to limited supplies of soluble iron in the soil. Iron deficiency induces a series of adaptive responses in various plant species, but the mechanisms by which they are triggered remain largely unknown. Using pH imaging and hormone localization techniques, it has been demonstrated here that root Fe(III) reductase activity and proton extrusion upon iron deficiency are up-regulated by systemic auxin signalling in a Fe-efficient woody plant, Malus xiaojinensis. Split-root experiments demonstrated that Fe-deprivation in a portion of the root system induced a dramatic increase in Fe(III) reductase activity and proton extrusion in the Fe-supplied portion, suggesting that the iron deficiency responses were mediated by a systemic signalling. Reciprocal grafting experiments of M. xiaojinensis with Malus baccata, a plant with no capability to produce the corresponding responses, indicate that the initiation of the systemic signalling is likely to be determined by roots rather than shoots. Iron deficiency induced a substantial increase in the IAA content in the shoot apex and supplying exogenous IAA analogues (NAA) to the shoot apex could mimic the iron deficiency to trigger the corresponding responses. Conversely, preventing IAA transport from shoot to roots blocked the iron deficiency responses. These results strongly indicate that the iron deficiency-induced physiological responses are mediated by systemic auxin signalling.

[Molecular mechanism for dynamic regulation of endogenous ABA signal level].

The process from stress signal perception and the trigger of ABA biosynthesis to dynamic regulation of ABA level is an important stress signaling pathway in cells. Compared to the downstream events in ABA signal transduction, the researches in this field are relatively lagged. Expression of synthase genes, such as ZEP in roots and rate-limiting enzyme genes NCED, AtRGS1 and ABA2, can be activated in response to stresses. However, the expression of genes encoding degradative enzymes, including 7'-, 8'-, 9'-hydroxylase and glucosyltransferase, negatively regulates ABA accumulation. Meanwhile, the expressions of the synthases, such as ZEP and NCED3, are induced by increasing endogenous ABA contents. Additionally, the analyses of gene expression and source-sink dynamics indicates that sustained supply from root-sourced ABA is required for the maintenance of leaf ABA dynamic pool. It is notable that miRNAs should be involved in ABA signal origin and ABA level dynamic adjustment. Further dynamic analysis of ABA metabolism revealed that endogenous ABA signal levels are synergistically controlled by the expressions of synthases and degradative enzymes.

Signal transduction in non-climacteric fruit ripening.

Fleshy fruit ripening involves changes in numerous cellular processes and metabolic pathways, resulting from the coordinated actions of diverse classes of structural and regulatory proteins. These include enzymes, transporters and complex signal transduction systems. Many aspects of the signaling machinery that orchestrates the ripening of climacteric fruits, such as tomato (Solanum lycopersicum), have been elucidated, but less is known about analogous processes in non-climacteric fruits. The latter include strawberry (Fragaria x ananassa) and grape (Vitis vinifera), both of which are used as non-climacteric fruit experimental model systems, although they originate from different organs: the grape berry is a true fruit derived from the ovary, while strawberry is an accessory fruit that is derived from the floral receptacle. In this article, we summarize insights into the signal transduction events involved in strawberry and grape berry ripening. We highlight the mechanisms underlying non-climacteric fruit ripening, the multiple primary signals and their integrated action, individual signaling components, pathways and their crosstalk, as well as the associated transcription factors and their signaling output.

Do Non-climacteric Fruits Share a Common Ripening Mechanism of Hormonal Regulation?

Fleshy fruits have been traditionally categorized into climacteric (CL) and non-climacteric (NC) groups. CL fruits share a common ripening mechanism of hormonal regulation, i.e., the ethylene regulation, whereas whether NC fruits share a common mechanism remains controversial. Abscisic acid (ABA) has been commonly thought to be a key regulator in NC fruit ripening; however, besides ABA, many other hormones have been increasingly suggested to play crucial roles in NC fruit ripening. NC fruits vary greatly in their organ origin, constitution, and structure. Development of different organs may be different in the pattern of hormonal regulation. It has been well demonstrated that the growth and development of strawberry, the model of NC fruits, is largely controlled by a hormonal communication between the achenes and receptacle; however, not all NC fruits contain achenes. Accordingly, it is particularly important to understand whether strawberry is indeed able to represent a universal mechanism for the hormonal regulation of NC fruit ripening. In this mini-review, we summarized the recent research advance on the hormone regulation of NC ripening in relation to fruit organ origination, constitution, and structure, whereby analyzing and discussing whether NC fruits may share a common mechanism of hormonal regulation.

Autocatalytic biosynthesis of abscisic acid and its synergistic action with auxin to regulate strawberry fruit ripening.

Abscisic acid (ABA) plays a major role in the regulation of strawberry fruit ripening; however, the origin of the ABA signal is largely unknown. Here, we report an autocatalytic mechanism for ABA biosynthesis and its synergistic interaction with the auxin to regulate strawberry fruit ripening. We demonstrate that ABA biosynthesis is self-induced in the achenes, but not in the receptacle, which results its substantial accumulation during ripening. ABA was found to regulate both IAA transport and biosynthesis, thereby modulating IAA content during both early fruit growth and later during ripening. Taken together, these results reveal the origins of the ABA signal and demonstrate the importance of its coordinated action with IAA in the regulation of strawberry fruit development and ripening.