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.

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.

OsMPK4 promotes phosphorylation and degradation of IPA1 in response to salt stress to confer salt tolerance in rice.

Salt stress adversely affects plant growth, development, and crop yield. Rice (Oryza sativa L.) is one of the most salt-sensitive cereal crops, especially at the early seedling stage. Mitogen-activated protein kinase (MAPK/MPK) cascades have been shown to play critical roles in salt response in Arabidopsis. However, the roles of the MPK cascade signaling in rice salt response and substrates of OsMPK remain largely unknown. Here, we report that the salt-induced OsMPK4-Ideal Plant Architecture 1 (IPA1) signaling pathway regulates the salt tolerance in rice. Under salt stress, OsMPK4 could interact with IPA1 and phosphorylate IPA1 at Thr180, leading to degradation of IPA1. Genetic evidence shows that IPA1 is a negative regulator of salt tolerance in rice, whereas OsMPK4 promotes salt response in an IPA1-dependent manner. Taken together, our results uncover an OsMPK4-IPA1 signal cascade that modulates the salt stress response in rice and sheds new light on the breeding of salt-tolerant rice varieties.

Chilling-induced phosphorylation of IPA1 by OsSAPK6 activates chilling tolerance responses in rice.

Chilling is a major abiotic stress harming rice development and productivity. The C-REPEAT BINDING FACTOR (CBF)-dependent transcriptional regulatory pathway plays a central role in cold stress and acclimation in Arabidopsis. In rice, several genes have been reported in conferring chilling tolerance, however, the chilling signaling in rice remains largely unknown. Here, we report the chilling-induced OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 6 (OsSAPK6)-IDEAL PLANT ARCHITECTURE 1 (IPA1)-OsCBF3 signal pathway in rice. Under chilling stress, OsSAPK6 could phosphorylate IPA1 and increase its stability. In turn, IPA1 could directly bind to the GTAC motif on the OsCBF3 promoter to elevate its expression. Genetic evidence showed that OsSAPK6, IPA1 and OsCBF3 were all positive regulators of rice chilling tolerance. The function of OsSAPK6 in chilling tolerance depended on IPA1, and overexpression of OsCBF3 could rescue the chilling-sensitive phenotype of ipa1 loss-of-function mutant. Moreover, the natural gain-of-function allele ipa1-2D could simultaneously enhance seedling chilling tolerance and increase grain yield. Taken together, our results revealed a chilling-induced OsSAPK6-IPA1-OsCBF signal cascade in rice, which shed new lights on chilling stress-tolerant rice breeding.

ζ-Carotene Isomerase Suppresses Tillering in Rice through the Coordinated Biosynthesis of Strigolactone and Abscisic Acid.

Rice tillering is an important agronomic trait affecting grain yield. Here, we identified a high-tillering mutant tillering20 (t20), which could be restored to the wild type by treatment with the strigolactone (SL) analog rac-GR24. T20 encodes a chloroplast ζ-carotene isomerase (Z-ISO), which is involved in the biosynthesis of carotenoids and their metabolites, SL and abscisic acid (ABA). The t20 mutant has reduced SL and ABA, raising the question of how SL and ABA biosynthesis is coordinated, and whether they have overlapping functions in tillering. We discovered that rac-GR24 stimulated T20 expression and enhanced all-trans-ß-carotene biosynthesis. Importantly, rac-GR24 also stimulated expression of Oryza sativa 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (OsNCED1) through induction of Oryza sativa HOMEOBOX12 (OsHOX12), promoting ABA biosynthesis in shoot base. On the other hand, ABA treatment significantly repressed SL biosynthesis and the ABA biosynthetic mutants displayed elevated SL biosynthesis. ABA treatment reduced the number of basal tillers in both t20 and wild-type plants. Furthermore, while ABA-deficient mutants aba1 and aba2 had the same number of basal tillers as wild type, they had more unproductive upper tillers at maturity. This work demonstrates complex interactions in the biosynthesis of carotenoid, SLs and ABA, and reveals a role for ABA in the regulation of rice tillering.

Two FERONIA-Like Receptor Kinases Regulate Apple Fruit Ripening by Modulating Ethylene Production.

Ethylene has long been known to be a critical signal controlling the ripening of climacteric fruits; however, the signaling mechanism underlying ethylene production during fruit development is unknown. Here, we report that two FERONIA-like receptor kinases (FERLs) regulate fruit ripening by modulating ethylene production in the climacteric fruit, apple (Malus×domestica). Bioinformatic analysis indicated that the apple genome contains 14 members of the FER family (MdFERL1-17), of these 17 FERLs, MdFERL6 was expressed at the highest level in fruit. Heterologous expression of MdFERL6 or MdFERL1, the apple homolog of Arabidopsis FER, in another climacteric fruit, tomato (Solanum lycopersicum) fruit delayed ripening and suppressed ethylene production. Overexpression and antisense expression of MdFERL6 in apple fruit calli inhibited and promoted ethylene production, respectively. Additionally, virus-induced gene silencing (VIGS) of SlFERL1, the tomato homolog of FER, promoted tomato fruit ripening and ethylene production. Both MdFERL6 and MdFERL1 physically interacted with MdSAMS (S-adenosylmethionine synthase), a key enzyme in the ethylene biosynthesis pathway. MdFERL6 was expressed at high levels during early fruit development, but dramatically declined when fruit ripening commenced, implying that MdFERL6 might limit ethylene production prior to fruit development and the ethylene production burst during fruit ripening. These results indicate that FERLs regulate apple and tomato fruit ripening, shedding light on the molecular mechanisms underlying ripening in climacteric fruit.

A FERONIA-Like Receptor Kinase Regulates Strawberry (Fragaria × ananassa) Fruit Ripening and Quality Formation.

Ripening of fleshy fruits is controlled by a series of intricate signaling processes. Here, we report a FERONIA/FER-like receptor kinase, FaMRLK47, that regulates both strawberry (Fragaria × ananassa) fruit ripening and quality formation. Overexpression and RNAi-mediated downregulation of FaMRLK47 delayed and accelerated fruit ripening, respectively. We showed that FaMRLK47 physically interacts with FaABI1, a negative regulator of abscisic acid (ABA) signaling, and demonstrated that FaMRLK47 regulates fruit ripening by modulating ABA signaling, a major pathway governing strawberry fruit ripening. In accordance with these findings, overexpression and RNAi-mediated downregulation of FaMRLK47 caused a decrease and increase, respectively, in the ABA-induced expression of a series of ripening-related genes. Additionally, overexpression and RNAi-mediated downregulation of FaMRLK47 resulted in an increase and decrease in sucrose content, respectively, as compared with control fruits, and respectively promoted and inhibited the expression of genes in the sucrose biosynthesis pathway (FaSS and FaSPS). Collectively, this study demonstrates that FaMRLK47 is an important regulator of strawberry fruit ripening and quality formation, and sheds light on the signaling mechanisms underlying strawberry fruit development and ripening.

Genome-Wide Identification and Expression Analysis of MRLK Family Genes Associated with Strawberry (Fragaria vesca) Fruit Ripening and Abiotic Stress Responses.

Malectin-like domain-containing receptor-like kinases (MRLK) constitute a large and divergent family of proteins in plants; however, little is known about the role of MRLKs in fruit growth and development. In this study, we characterized MRLK family genes in diploid strawberry, Fragaria vesca. Based on an analysis of malectin-like domain and a search in the strawberry genome and NCBI database, we identified 62 FvMRLKs in the strawberry genome, and classified these genes into six subfamilies with distinct malectin domains in the extracellular regions of the encoded proteins. Gene expression analysis indicated that more than 80% of the FvMRLKs were expressed in various tissues, with higher levels in roots than in other organs. Thirty-three FvMRLKs were found to be expressed in fruits during the early stages of development, and over 60% of these exhibited dramatic decreases in expression during fruit growth and development. Moreover, the expression of some FvMRLKs was sensitive to both environmental and internal cues that play critical roles in regulating strawberry fruit development and ripening. Collectively, this study provides valuable insight into the FvMRLKs gene family and its role in regulating strawberry fruit development and ripening.