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Ubiquitination is an important post-translational protein modification. Although BROAD-COMPLEX, TRAMTRACK AND BRIC A BRAC and TRANSCRIPTION ADAPTOR PUTATIVE ZINC FINGER domain protein 2 (BT2) is involved in many biological processes, its role in apple (Malus domestic) root formation remains unclear. Here, we revealed that MdBT2 inhibits adventitious root (AR) formation through interacting with AUXIN RESPONSE FACTOR8 (MdARF8) and INDOLE-3-ACETIC ACID INDUCIBLE3 (MdIAA3). MdBT2 facilitated MdARF8 ubiquitination and degradation through the 26S proteasome pathway and negatively regulated GRETCHEN HAGEN 3.1 (MdGH3.1) and MdGH3.6 expression. MdARF8 regulates AR formation through inducing transcription of MdGH3s (MdGH3.1, MdGH3.2, MdGH3.5, and MdGH3.6). In addition, MdBT2 facilitated MdIAA3 stability and slightly promoted its interaction with MdARF8. MdIAA3 inhibited AR formation by forming heterodimers with MdARF8 as well as other MdARFs (MdARF5, MdARF6, MdARF7, and MdARF19). Our findings reveal that MdBT2 acts as a negative regulator of AR formation in apple.

Less than 40% of the nitrogen (N) fertilizer applied to soil is absorbed by crops. Thus, improving the N use efficiency of crops is critical for agricultural development. However, the underlying regulation of these processes remains largely unknown, particularly in woody plants. By conducting yeast two-hybrid assays, we identified one interacting protein of MdMYB88 and MdMYB124 in apple (Malus × domestica), namely BTB and TAZ domain protein 2 (MdBT2). Ubiquitination and protein stabilization analysis revealed that MdBT2 ubiquitinates and degrades MdMYB88 and MdMYB124 via the 26S proteasome pathway. MdBT2 negatively regulates nitrogen usage as revealed by the reduced fresh weight, dry weight, N concentration, and N usage index of MdBT2 overexpression calli under low-N conditions. In contrast, MdMYB88 and MdMYB124 increase nitrate absorption, allocation, and remobilization by regulating expression of MdNRT2.4, MdNRT1.8, MdNRT1.7, and MdNRT1.5 under N limitation, thereby regulating N usage. The results obtained illustrate the mechanism of a regulatory module comprising MdBT2–MdMYB88/MdMYB124–MdNRTs, through which plants modulate N usage. These data contribute to a molecular approach to improve the N usage of fruit crops under limited N acquisition.

The ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) plays a central role in light-induced anthocyanin biosynthesis. However, the upstream regulatory factors of COP1 remain poorly understood, particularly in horticultural plants. Here, we identified an MdCOP1-interacting protein, BROAD-COMPLEX, TRAMTRACK AND BRIC A BRAC2 (MdBT2), in apple (Malus domestica). MdBT2 is a BTB protein that directly interacts with and stabilizes MdCOP1 by inhibiting self-ubiquitination. Fluorescence observation and cell fractionation assays showed that MdBT2 increased the abundance of MdCOP1 in the nucleus. Moreover, a series of phenotypic analyses indicated that MdBT2 promoted MdCOP1-mediated ubiquitination and degradation of the MdMYB1 transcription factor, inhibiting the expression of anthocyanin biosynthesis genes and anthocyanin accumulation. Overall, our findings reveal a molecular mechanism by which MdBT2 positively regulates MdCOP1, providing insight into MdCOP1-mediated anthocyanin biosynthesis.

Excessive application of nitrate, an essential macronutrient and a signal regulating diverse physiological processes, decreases malate accumulation in apple (Malus domestica) fruit, but the underlying mechanism remains poorly understood. Here, we show that an apple BTB/TAZ protein, MdBT2, is involved in regulating malate accumulation and vacuolar pH in response to nitrate. In vitro and in vivo assays indicate that MdBT2 interacts directly with and ubiquitinates a bHLH transcription factor, MdCIbHLH1, via the ubiquitin/26S proteasome pathway in response to nitrate. This ubiquitination results in the degradation of MdCIbHLH1 protein and reduces the transcription of MdCIbHLH1-targeted genes involved in malate accumulation and vacuolar acidification, including MdVHA-A, which encodes a vacuolar H+-ATPase, and MdVHP1, which encodes a vacuolar H+-pyrophosphatase, as well as MdALMT9, which encodes an aluminum-activated malate transporter. A series of transgenic analyses in apple materials including fruits, plantlets, and calli demonstrate that MdBT2 controls nitrate-mediated malate accumulation and vacuolar pH at least partially, if not completely, via regulating the MdCIbHLH1 protein level. Taken together, these findings reveal that MdBT2 regulates the stability of MdCIbHLH1 via ubiquitination in response to nitrate, which in succession transcriptionally reduces the expression of malate-associated genes, thereby controlling malate accumulation and vacuolar acidification in apples under high nitrate supply.

Nitrate acts as a vital signal molecule in the modulation of plant growth and development. The phytohormones gibberellin (GA) is also involved in this process. However, the exact molecular mechanism of how nitrate and GA signaling pathway work together in regulating plant growth remains poorly understood. In this study, we found that a nitrate-responsive BTB/TAZ protein MdBT2 participates in regulating nitrate-induced plant growth in apple (Malus × domestica). Yeast two-hybridization, protein pull-down, and bimolecular fluorescence complementation (BiFC) assays showed that MdBT2 interacts with a DELLA protein MdRGL3a, which is required for the ubiquitination and degradation of MdRGL3a proteins via a 26S proteasome-dependent pathway. Furthermore, heterologous expression of MdBT2 partially rescued growth inhibition caused by overexpression of MdRGL3a in Arabidopsis. Taken together, our findings indicate that MdBT2 promotes nitrate-induced plant growth partially through reducing the abundance of the DELLA protein MdRGL3a.

SIZ1 (a SIZ/PIAS-type SUMO E3 ligase)-mediated small ubiquitin-like modifier (SUMO) modification of target proteins is important for various biological processes related to abiotic stress resistance in plants; however, little is known about its role in resistance toward iron (Fe) deficiency. Here, the SUMO E3 ligase MdSIZ1 was shown to be involved in the plasma membrane (PM) H+-ATPase-mediated response to Fe deficiency. Subsequently, a basic helix-loop-helix transcription factor, MdbHLH104 (a homolog of Arabidopsis bHLH104 in apple), which acts as a key component in regulating PM H+-ATPase-mediated rhizosphere acidification and Fe uptake in apples (Malus domestica), was identified as a direct target of MdSIZ1. MdSIZ1 directly sumoylated MdbHLH104 both in vitro and in vivo, especially under conditions of Fe deficiency, and this sumoylation was required for MdbHLH104 protein stability. Double substitution of K139R and K153R in MdbHLH104 blocked MdSIZ1-mediated sumoylation in vitro and in vivo, indicating that the K139 and K153 residues were the principal sites of SUMO conjugation. Moreover, the transcript level of the MdSIZ1 gene was substantially induced following Fe deficiency. MdSIZ1 overexpression exerted a positive influence on PM H+-ATPase-mediated rhizosphere acidification and Fe uptake. Our findings reveal an important role for sumoylation in the regulation of PM H+-ATPase-mediated rhizosphere acidification and Fe uptake during Fe deficiency in plants.

In addition to scavenging reactive oxygen species, anthocyanins are pigments that give organs their color. In apple (Malus domestica), R2R3-MYB transcription factor MdMYB1 is a master regulator of anthocyanin biosynthesis and fruit coloration. In this study, we found that MdMYB1 was degraded via a ubiquitin-dependent pathway in response to nitrate, an inhibitor of anthocyanin synthesis. Using a yeast two-hybrid (Y2H) approach, we found that the BTB-TAZ protein encoded by the nitrate-responsive gene MdBT2 interacts with MdMYB1. Pull-down and coimmunoprecipitation assays supported this conclusion. In vivo and in vitro experiments revealed that MdBT2 promoted the ubiquitination and degradation of MdMYB1 through a cullin protein MdCUL3-independent pathway. Expression analysis demonstrated that MdBT2 and MdMYB1 were inversely regulated by nitrate and other environmental signals. Furthermore, we used transgenic approaches in apple and Arabidopsis (Arabidopsis thaliana) to characterize the function of MdBT2 in regulating anthocyanin biosynthesis in response to nitrate. Our findings provide insight into a mechanism involving the MdBT2-MdMYB1 pathway that regulates anthocyanin accumulation in apple and possibly in other plant species.

Ethylene regulates climacteric fruit ripening, and EIN3-LIKE1 (EIL1) plays an important role in this process. In apple (Malus domestica), fruit coloration is accompanied by ethylene release during fruit ripening, but the molecular mechanism that underlies these two physiological processes is unknown. In this study, we found that ethylene treatment markedly induced fruit coloration as well as the expression of MdMYB1, a positive regulator of anthocyanin biosynthesis and fruit coloration. In addition, we found that MdEIL1 directly bound to the promoter of MdMYB1 and transcriptionally activated its expression, which resulted in anthocyanin biosynthesis and fruit coloration. Furthermore, MdMYB1 interacted with the promoter of ETHYLENE RESPONSE FACTOR3, a key regulator of ethylene biosynthesis, thereby providing a positive feedback for ethylene biosynthesis regulation. Overall, our findings provide insight into a mechanism involving the synergistic interaction of the ethylene signal with the MdMYB1 transcription factor to regulate ethylene biosynthesis and fruit coloration in apple.

Glc regulates many vital processes, including plant growth, development, metabolism, and responses to biotic and abiotic stress. However, the molecular mechanism by which Glc acts as a signal to regulate salinity tolerance remains unclear. In this study, we found that the apple (Malus domestica Borkh.) Glc sensor hexokinase1 (MdHXK1) contributes to Glc-mediated salinity tolerance. A combination of split ubiquitin system, pull-down, co-immunoprecipitation, and bimolecular fluorescence complementation assays demonstrated that MdHXK1 interacts with and phosphorylates the Na+/H+ exchanger MdNHX1 at its Ser-275 residue. Phosphorylation improved the stability of MdNHX1 and enhanced its Na+/H+ transport activity in MdNHX1 overexpression transgenic apple and yeast complementation cells. Furthermore, Ser-275 of MdNHX1 was found to be crucial for MdHXK1-mediated phosphorylation. Finally, a series of transgenic analyses demonstrated that salt tolerance mediated by MdHXK1 partially depended on MdNHX1. Overall, our findings provide insights into how sugar recruits and regulates MdNHX1 in response to high salinity in plants.

Carotenoids exert multifaceted roles to plants and are critically important to humans. Phytoene synthase (PSY) is a major rate-limiting enzyme in the carotenoid biosynthetic pathway. PSY in plants is normally found as a small enzyme family with up to three members. However, knowledge of PSY isoforms in relation to their respective enzyme activities and amino acid residues that are important for PSY activity is limited. In this study, we focused on two tomato (Solanum lycopersicum) PSY isoforms, PSY1 and PSY2, and investigated their abilities to catalyze carotenogenesis via heterologous expression in transgenic Arabidopsis (Arabidopsis thaliana) and bacterial systems. We found that the fruit-specific PSY1 was less effective in promoting carotenoid biosynthesis than the green tissue–specific PSY2. Examination of the PSY proteins by site-directed mutagenesis analysis and three-dimensional structure modeling revealed two key amino acid residues responsible for this activity difference and identified a neighboring aromatic-aromatic combination in one of the PSY core structures as being crucial for high PSY activity. Remarkably, this neighboring aromatic-aromatic combination is evolutionarily conserved among land plant PSYs except PSY1 of tomato and potato (Solanum tuberosum). Strong transcription of tomato PSY1 likely evolved as compensation for its weak enzyme activity to allow for the massive carotenoid biosynthesis in ripe fruit. This study provides insights into the functional divergence of PSY isoforms and highlights the potential to rationally design PSY for the effective development of carotenoid-enriched crops.

Iron (Fe) homeostasis is crucial for plant growth and development. A network of basic helix-loop-helix (bHLH) transcription factors positively regulates Fe uptake during iron deficiency. However, their up-regulation or overexpression leads to Fe overload and reactive oxygen species generation, thereby damaging the plants. Here, we found that two BTB/TAZ proteins, MdBT1 and MdBT2, interact with the MbHLH104 protein in apple. In addition, the function of MdBT2 was characterized as a regulator of MdbHLH104 degradation via ubiquitination and the 26S proteasome pathway, thereby controlling the activity of plasma membrane H+-ATPases and the acquisition of iron. Furthermore, MdBT2 interacted with MdCUL3 proteins, which were required for the MdBT2-mediated ubiquitination modification of MdbHLH104 and its degradation. In sum, our findings demonstrate that MdBT proteins interact with MdCUL3 to bridge the formation of the MdBTsMdCUL3 complex, which negatively modulates the degradation of the MdbHLH104 protein in response to changes in Fe status to maintain iron homeostasis in plants.

MdMYB1 is a crucial regulator of light-induced anthocyanin biosynthesis and fruit coloration in apple (Malus domestica). In this study, it was found that MdMYB1 protein accumulated in the light but degraded via a ubiquitin-dependent pathway in the dark. Subsequently, the MdCOP1-1 and MdCOP1-2 genes were isolated from apple fruit peel and were functionally characterized in the Arabidopsis (Arabidopsis thaliana) cop1-4 mutant. Yeast (Saccharomyces cerevisiae) two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays showed that MdMYB1 interacts with the MdCOP1 proteins. Furthermore, in vitro and in vivo experiments indicated that MdCOP1s are necessary for the ubiquitination and degradation of MdMYB1 protein in the dark and are therefore involved in the light-controlled stability of the MdMYB1 protein. Finally, a viral vector-based transformation approach demonstrated that MdCOP1s negatively regulate the peel coloration of apple fruits by modulating the degradation of the MdMYB1 protein. Our findings provide new insight into the mechanism by which light controls anthocyanin accumulation and red fruit coloration in apple and even other plant species.

Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H(+)-pumping activities of vacuolar H(+)-ATPase (VHA) and/or vacuolar H(+)-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H(+)-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants.

Optically active forms of abscisic acid (ABA) and their oxygenated metabolites were tested for their biological activity by examining the effects of the compounds on the reversal of gibberellic acid-induced [alpha]-amylase activity in barley (Hordeum vulgare cv Himalaya) aleurone layers and the induction of gene expression in barley aleurone protoplasts transformed with a chimeric construct containing the promoter region of an albumin storage protein gene. Promotion of the albumin storage protein gene response had a more strict stereochemical requirement for elicitation of an ABA response than inhibition of [alpha]-amylase gene expression. The naturally occurring stereoisomer of ABA and its metabolites were more effective at eliciting an ABA-like response. ABA showed the highest activity, followed by 7[prime]-hydroxyABA, with phaseic acid being the least active. Racemic 8[prime]-hydroxy-2[prime],3[prime]-dihydroABA, an analog of 8[prime]-hydroxyABA, was inactive, whereas racemic 2[prime],3[prime]-dihydroABA was as effective as ABA. The differences in response of the same tissue to the ABA enantiomers lead us to conclude that there exists more than one type of ABA receptor and/or multiple signal transduction pathways in barley aleurone tissue.