The BTB protein MdBT2 recruits auxin signaling components to regulate adventitious root formation in apple.

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.

Low nitrate alleviates iron deficiency by regulating iron homeostasis in apple.

Iron (Fe) is an essential element for plant growth, development and metabolism. Due to its lack of solubility and low bioavailability in soil, Fe levels are usually far below the optimum amount for most plants' growth and development. In apple production, excessive use of nitrogen fertilizer may cause iron chlorosis symptoms in the newly growing leaves, but the regulatory mechanisms underlying this phenomenon are unclear. In this study, low nitrate (NO3- , LN) application alleviated the symptoms of Fe deficiency and promoted lower rhizosphere pH, which was beneficial for root Fe acquisition. At the same time, LN treatment increased citrate and abscisic acid accumulation in roots, which promoted Fe transport from root to shoot and maintained Fe homeostasis. Moreover, qRT-PCR analysis showed that nitrate application caused differential expression of genes related to Fe uptake and transport, as well as transcriptional regulators. In summary, our data reveal that low nitrate alleviated Fe deficiency through multiple pathways, demonstrating a new option for minimizing Fe deficiency by regulating the balance between nutrients.

Auxin regulates anthocyanin biosynthesis through the auxin repressor protein MdIAA26.

Auxin plays an important role in plant growth and development; for example, it regulates the elongation and division of plant cells, the formation of plantlet's geotropism and phototropism, and the growth of main lateral roots and hypocotyl. IAA gene is associated with auxin and can response to biotic and abiotic stress in plants. However, the regulatory effect of auxin on anthocyanin accumulation has been rarely reported. In this study, we show that auxin inhibites the accumulation of anthocyanin and decreases the expression of genes related to anthocyanin synthesis in calli, leaves, and seedlings of apple. The expression levels of MdIAA family genes were determined, and we found that MdIAA26 significantly responded to auxin, which also induced MdIAA26 degradation. Functional analysis of MdIAA26 showed that overexpressing MdIAA26 in apple calli and Arabidopsis could promote the accumulation of anthocyanin and up-regulate the genes related to anthocyanin synthesis. Furthermore, the MdIAA26-overexpressing Arabidopsis could counteract auxin-induced inhibition on anthocyanin accumulation, which indicates that auxin inhibits the accumulation of anthocyanin in apple by degrading MdIAA26 protein.

The apple C2H2-type zinc finger transcription factor MdZAT10 positively regulates JA-induced leaf senescence by interacting with MdBT2.

Jasmonic acid (JA) plays an important role in regulating leaf senescence. However, the molecular mechanisms of leaf senescence in apple (Malus domestica) remain elusive. In this study, we found that MdZAT10, a C2H2-type zinc finger transcription factor (TF) in apple, markedly accelerates leaf senescence and increases the expression of senescence-related genes. To explore how MdZAT10 promotes leaf senescence, we carried out liquid chromatography/mass spectrometry screening. We found that MdABI5 physically interacts with MdZAT10. MdABI5, an important positive regulator of leaf senescence, significantly accelerated leaf senescence in apple. MdZAT10 was found to enhance the transcriptional activity of MdABI5 for MdNYC1 and MdNYE1, thus accelerating leaf senescence. In addition, we found that MdZAT10 expression was induced by methyl jasmonate (MeJA), which accelerated JA-induced leaf senescence. We also found that the JA-responsive protein MdBT2 directly interacts with MdZAT10 and reduces its protein stability through ubiquitination and degradation, thereby delaying MdZAT10-mediated leaf senescence. Taken together, our results provide new insight into the mechanisms by which MdZAT10 positively regulates JA-induced leaf senescence in apple.

Genome-Wide Identification of Apple Ubiquitin SINA E3 Ligase and Functional Characterization of MdSINA2.

SINA (Seven in absentia) proteins are a small family of ubiquitin ligases that play important roles in regulating plant growth and developmental processes as well as in responses to diverse types of biotic and abiotic stress. However, the characteristics of the apple SINA family have not been previously studied. Here, we identified 11 MdSINAs members in the apple genome based on their conserved, N-terminal RING and C-terminal SINA domains. We also reconstructed a phylogeny of these genes; characterized their chromosomal location, structure, and motifs; and identified two major groups of MdSINA genes. Subsequent qRT-PCR analyses were used to characterize the expression of MdSINA genes in various tissues and organs, and levels of expression were highest in leaves. MdSINAs were significantly induced under ABA and carbon- and nitrate-starvation treatment. Except for MdSINA1 and MdSINA7, the other MdSINA proteins could interact with each other. Moreover, MdSINA2 was found to be localized in the nucleus using Agrobacterium-mediated transient expression. Western-blot analysis showed that MdSINA2 accumulated extensively under light, decreased under darkness, and became insensitive to light when the RING domain was disrupted. Finally, ABA-hypersensitive phenotypes were confirmed by transgenic calli and the ectopic expression of MdSINA2 in Arabidopsis. In conclusion, our results suggest that MdSINA genes participate in the responses to different types of stress, and that MdSINA2 might act as a negative regulator in the ABA stress response.

The BTB-TAZ protein MdBT2 negatively regulates the drought stress response by interacting with the transcription factor MdNAC143 in apple.

Drought stress is a severe source of abiotic stress that can affect apple yield and quality, yet the underlying molecular mechanism of the drought stress response and the role of MdBT2 in the process remain unclear. Here, we find that MdBT2 negatively regulates the drought stress response. Both in vivo and in vitro assays indicated that MdBT2 interacted physically with and ubiquitinated MdNAC143, a member of the NAC TF family that is a positive regulator under drought stress. In addition, MdBT2 promotes the degradation of MdNAC143 via the 26S proteasome system. A series of transgenic assays in apple calli and Arabidopsis verify that MdBT2 confers susceptibility to drought stress at least in part by the regulation of MdNAC143. Overall, our findings provide new insight into the mechanism of MdBT2, which functions antagonistically to MdNAC143 in regulating drought stress by regulating the potential downstream target protein MdNAC143 for proteasomal degradation in apple.

Phosphate regulates malate/citrate-mediated iron uptake and transport in apple.

The accumulation of iron (Fe) in the apical meristem is considered as a critical factor involved in limiting the elongation of roots under low phosphate (Pi) conditions. Furthermore, the antagonism between Fe and Pi largely affects the effective utilization of Fe. Although the lack of Pi serves to increase the effectiveness of Fe in rice under both Fe-sufficient and Fe-deficient conditions, the underlying physiological mechanism governing this phenomenon is still unclear. In this study, we found that low Pi alleviated the Fe-deficiency phenotype in apples. Additionally, low Pi treatments increased ferric-chelated reductase (FCR) activity in the rhizosphere, promoted proton exocytosis, and enhanced the Fe concentration in both the roots and shoots. In contrast, high Pi treatments inhibited this process. Under conditions of low Pi, malate and citrate exudation from apple roots occurred under both Fe-sufficient and Fe-deficient conditions. In addition, treatment with 0.5 mM malate and citrate effectively alleviated the Fe and Pi deficiencies. Taken together, these data support the conclusion that a low Pi supply promotes organic acids exudation and enhances Fe absorption during Fe deficiency in apples.

Identification of a Novel Alternative Splicing Variant of VvPMA1 in Grape Root under Salinity.

It has been well-demonstrated that the control of plasma membrane H+-ATPase (PM H+-ATPase) activity is important to plant salt tolerance. This study found a significant increase in PM H+-ATPase (PMA) activity in grape root exposed to NaCl. Furthermore, 7 Vitis vinifera PM H+-ATPase genes (VvPMAs) were identified within the grape genome and the expression response of these VvPMAs in grape root under salinity was analyzed. Two VvPMAs (VvPMA1 and VvPMA3) were expressed more strongly in roots than the other five VvPMAs. Moreover, roots exhibited diverse patterns of gene expression of VvPMA1 and VvPMA3 responses to salt stress. Interestingly, two transcripts of VvPMA1, which were created through alternative splicing (AS), were discovered and isolated from salt stressed root. Comparing the two VvPMA1 cDNA sequences (designated VvPMA1α and VvPMA1ß) with the genomic sequence revealed that the second intron was retained in the VvPMA1ß cDNA. This intron retention was predicted to generate a novel VvPMA1 through N-terminal truncation because of a 5'- terminal frame shift. Yeast complementation assays of the two splice variants showed that VvPMA1ß could enhance the ability to complement Saccharomyces cerevisiae deficient in PM H+-ATPase activity. In addition, the expression profiles of VvPMA1α and VvPMA1ß differed under salinity. Our data suggests that through AS, the N-terminal length of VvPMA1 may be regulated to accurately modulate PM H+-ATPase activity of grape root in salt stress.