SlPti4 Affects Regulation of Fruit Ripening, Seed Germination and Stress Responses by Modulating ABA Signaling in Tomato.

Although the role of the ethylene response factor (ERF) Pti4 in disease resistance has been demonstrated in higher plants, it is presently unknown whether the tomato SlPti4 protein plays a role in the regulation of fruit development and the stress response. Here, we show that SlPti4 is involved in the regulation of fruit ripening, seed germination, and responses to drought and Botrytis cinerea infection through adjustments to ABA metabolism and signaling. SlPti4 gene expression is very low early in fruit development, but increases rapidly during ripening and can be induced by exogenous ABA and 1-aminocyclopropane 1-carboxylate (ACC). RNA interference (RNAi)-induced silencing of SlPti4 leads to an increase of ABA accumulation together with a decrease of ethylene release, which causes the high expression level of SlBcyc, and thus the transgenic fruit is orange instead of red as in wild-type fruit during ripening. SlPti4-RNAi seeds accumulate less ABA and mRNA for ABA receptor SlPYL genes, which causes insensitivity to ABA treatment. SlPti4-RNAi transgenic plants with low ABA levels and high ethylene release were more sensitive to drought stress. SlPti4-RNAi plants also showed weaker resistance to B. cinerea infection than the wild type. Thus, SlPti4 is an important regulator of tomato fruit ripening, seed germination and abiotic/biotic stress responses. This study expands our knowledge on diverse plant physiologies which are regulated by ABA signaling and the function of SlPti4.

The functional analysis of SlNCED1 in tomato pollen development.

Abscisic acid (ABA) regulates plant growth and development, but the role of ABA in the development of reproductive organs in tomato has rarely been addressed. In the present study, the role of ABA in the regulation of male and female gametogenesis as well as pollen development and germination is tested in tomato. qRT-PCR and in situ hybridization analysis of 9-cis-epoxycarotenoid dioxygenase (SlNCED1), a key enzyme in the ABA biosynthetic pathway, showed high expression of SlNCED1 primarily in the meristem during gametogenesis and mainly in ovule, stigma, anther/pollen and vascular tissues during floral organ development. SlNCED1 expression and ABA accumulation in anther peak at stages 13-14, suggesting that ABA plays a role in the primary formation of pollen grains. Over expression and suppression of SlNCED1 led to the abnormal development of anther/pollen, especially in SlNCED1-OE lines, which have serious pollen deterioration. The percentage of pollen germination in wild type is 91.47%, whereas it is 6.85% in OE transgenic lines and 38.4% at anthesis in RNAi lines. RNA-Seq of anthers shows that SlNCED1-OE can significantly enhance the expression of SlPP2Cs and down-regulate the expression of SlMYB108 and SlMYB21, which are anther/flower-specific transcriptional factors in tomato. Finally, anther transcriptome data indicate that SlNCED1 is involved in ABA-mediated regulation in pollen/anther metabolism, cell wall modification, and transcription levels. These results support an important role for ABA in the development of reproductive organs in tomato and contribute to the elucidation of the underlying regulatory mechanisms.

Suppressing Type 2C Protein Phosphatases Alters Fruit Ripening and the Stress Response in Tomato.

Although ABA signaling has been widely studied in Arabidopsis, the roles of core ABA signaling components in fruit remain poorly understood. Herein, we characterize SlPP2C1, a group A type 2C protein phosphatase that negatively regulates ABA signaling and fruit ripening in tomato. The SlPP2C1 protein was localized in the cytoplasm close to AtAHG3/AtPP2CA. The SlPP2C1 gene was expressed in all tomato tissues throughout development, particularly in flowers and fruits, and it was up-regulated by dehydration and ABA treatment. SlPP2C1 expression in fruits was increased at 30 d after full bloom and peaked at the B + 1 stage. Suppression of SlPP2C1 expression significantly accelerated fruit ripening which was associated with higher levels of ABA signaling genes that are reported to alter the expression of fruit ripening genes involved in ethylene release and cell wall catabolism. SlPP2C1-RNAi (RNA interference) led to increased endogenous ABA accumulation and advanced release of ethylene in transgenic fruits compared with wild-type (WT) fruits. SlPP2C1-RNAi also resulted in abnormal flowers and obstructed the normal abscission of pedicels. SlPP2C1-RNAi plants were hypersensitized to ABA, and displayed delayed seed germination and primary root growth, and increased resistance to drought stress compared with WT plants. These results demonstrated that SlPP2C1 is a functional component in the ABA signaling pathway which participates in fruit ripening, ABA responses and drought tolerance.

Suppressing ABA uridine diphosphate glucosyltransferase (SlUGT75C1) alters fruit ripening and the stress response in tomato.

Abscisic acid (ABA) glucose conjugation mediated by uridine diphosphate glucosyltransferases (UGTs) is an important pathway in regulating ABA homeostasis. In the present study, we investigated three tomato SlUGTs that are highly expressed in fruit during ripening, and these SlUGTs were localized to the cytoplasm and cell nucleus. Among these three UGTs, SlUGT75C1 catalyzes the glucosylation of both ABA and IAA in vitro; SlUGT76E1 can only catalyze the conjugation of ABA; and SlUGT73C4 cannot glycosylate either ABA or IAA. Therefore, SlUGT75C1 was selected for further investigation. SlUGT75C1 RNA interference significantly up-regulated the expression level of SlCYP707A2, which encodes an ABA 8'-hydroxylase but did not affect the expression of SlNCED1, which encodes a key enzyme in ABA biosynthesis. Suppression of SlUGT75C1 significantly accelerated fruit ripening by enhancing ABA levels and promoting the early release of ethylene. SlUGT75C1-RNAi altered the expression of fruit ripening genes (genes involved in ethylene release and cell wall catabolism). SlUGT75C1-RNAi seeds showed delayed germination and root growth compared with wild-type as well as increased sensitivity to exogenous ABA. SlUGT75C1-RNAi plants were also more resistant to drought stress. These results demonstrated that SlUGT75C1 plays a crucial role in ABA-mediated fruit ripening, seed germination, and drought responses in tomato.

Variable responses of two VlMYBA gene promoters to ABA and ACC in Kyoho grape berries.

The VlMYBA subfamily of transcription factors has been known to be the functional regulators in anthocyanin biosynthesis in red grapes. In this study, the expressions of the VlMYBA1-2 and VlMYBA 2 genes, and the responses of the VlMYBA1-2/2 promoters to ABA and ACC treatments in Kyoho grape berries are examined through quantitative real-time PCR analysis and the transient expression assay. The results show that the expressions of VlMYBA1-2/2 increase dramatically after véraison and reach their highest levels when the berries are nearly fully ripe. Exogenous ABA promotes the expressions of VlMYBA1-2/2, whereas the ACC treatment increases the expression of VlMYBA2, however, it has no effect on VlMYBA1-2. The ABA treatment has a faster and stronger effect on berry pigmentation than ACC does. The VlMYBA1-2 promoter sequence contains two ABA response elements (ABRE) but no ethylene response element (ERE), whereas the VlMYBA2 promoter sequence contains two ABRE and one ERE in the upstream region of the start codon. The VlMYBA2 promoter can be activated by both ABA (more effective) and ACC, whereas the VlMYBA1-2 promoter can be activated by ABA only. In sum, ABA can promote the coloring of Kyoho grape by the promotion of VlMYBA1-2/2 transcriptions via activating the response of their promoters to ABA, whereas ethylene only regulates VlMYBA2 through the response activation of its promoter to ACC which partially enhances the coloring.

Interactions of ABA signaling core components (SlPYLs, SlPP2Cs, and SlSnRK2s) in tomato (Solanum lycopersicon).

Abscisic acid (ABA) regulates fruit development and ripening via its signaling. However, the exact role of ABA signaling core components in fruit have not yet been clarified. In this study, we investigated the potential interactions of tomato (Solanum lycopersicon) ABA signaling core components using yeast two-hybrid analysis, with or without ABA at different concentrations. The results showed that among 12 PYR/PYL/RCAR ABA receptors (SlPYLs), SlPYL1, SlPYL2, SlPYL4, SlPYL5, SlPYL 7, SlPYL8, SlPYL9, SlPYL10, SlPYL11, and SlPYL13 were ABA-dependent receptors, while SlPYL3 and SlPYL12 were ABA-independent receptors. Among five SlPP2Cs (type 2C protein phosphatases) and seven SlSnRK2s (subfamily 2 of SNF1-related kinases), all SlSnRK2s could interact with SlPP2C2, while SlSnRK2.8 also interacted with SlPP2C3. SlSnRK2.5 could interact with SlABF2/4 (ABA-responsive element binding factors). Expressions of SlPYL1, SlPYL2, SlPYL8, and SlPYL10 were upregulated under exogenous ABA but downregulated under nordihydroguaiaretic acid (NDGA) at the mature green stage of fruit ripening. The expressions of SlPP2C1, SlPP2C2, SlPP2C3, and SlPP2C5 were upregulated in ABA-treated fruit, but downregulated in NDGA-treated fruit at the mature green stage. The expressions of SlSnRK2.4, SlSnRK2.5, SlSnRK2.6, and SlSnRK2.7 were upregulated by ABA, but downregulated by NDGA. However, SlSnRK2.2 was down regulated by ABA. Expression of SlABF2/3/4 was enhanced by ABA but decreased by NDGA. Based on these results, we concluded that the majority of ABA receptor PYLs interact with SlPP2Cs in an ABA-dependent manner. SlPP2C2 and SlPP2C3 can interact with SlSnRK2s. SlSnRK2.5 could interact with SlABF2/4. Most ABA signaling core components respond to exogenous ABA.