Dissection of mRNA ac4C acetylation modifications in AC and Nr fruits: insights into the regulation of fruit ripening by ethylene.

N4-acetylcytidine (ac4C) modification of mRNA has been shown to be present in plant RNAs, but its regulatory function in plant remains largely unexplored. In this study, we investigated the differentially expressed mRNAs, lncRNAs and acetylation modifications of mRNAs in tomato fruits from both genotypes. By comparing wild-type (AC) tomato and the ethylene receptor-mutant (Nr) tomato from mature green (MG) to six days after the breaker (Br6) stage, we identified differences in numerous key genes related to fruit ripening and observed the corresponding lncRNAs positively regulated the target genes expression. At the post-transcriptional level, the acetylation level decreased and increased in AC and Nr tomatoes from MG to Br6 stage, respectively. The integrated analysis of RNA-seq and ac4C-seq data revealed the potential positive role of acetylation modification in regulating gene expression. Furthermore, we found differential acetylation modifications of certain transcripts (ACO, ETR, ERF, PG, CesA, ß-Gal, GAD, AMY, and SUS) in AC and Nr fruits which may explain the differences in ethylene production, fruit texture, and flavor during their ripening processes. The present study provides new insights into the molecular mechanisms by which acetylation modification differentially regulates the ripening process of wild-type and mutant tomato fruits deficient in ethylene signaling.

Decoding plant adaptation: deubiquitinating enzymes UBP12 and UBP13 in hormone signaling, light response, and developmental processes.

Ubiquitination, a vital post-translational modification in plants, plays a significant role in regulating protein activity, localization, and stability. This process occurs through a complex enzyme cascade that involves E1, E2, and E3 enzymes, leading to the covalent attachment of ubiquitin molecules to substrate proteins. Conversely, deubiquitinating enzymes (DUBs) work in opposition to this process by removing ubiquitin moieties. Despite extensive research on ubiquitination in plants, our understanding of the function of DUBs is still emerging. UBP12 and UBP13, two plant DUBs, have received much attention recently and are shown to play pivotal roles in hormone signaling, light perception, photoperiod responses, leaf development, senescence, and epigenetic transcriptional regulation. This review summarizes current knowledge of these two enzymes, highlighting the central role of deubiquitination in regulating the abundance and activity of critical regulators such as receptor kinases and transcription factors during phytohormone and developmental signaling.

The kiwifruit amyloplast proteome (kfALP): a resource to better understand the mechanisms underlying amyloplast biogenesis and differentiation.

The biogenesis and differentiation (B&D) of amyloplasts contributes to fruit flavor and color. Here, remodeling of starch granules, thylakoids and plastoglobules was observed during development and ripening in two kiwifruit (Actinidia spp.) cultivars - yellow-fleshed 'Hort16A' and green-fleshed 'Hayward'. A protocol was developed to purify starch-containing plastids with a high degree of intactness, and amyloplast B&D was studied using label-free-based quantitative proteomic analyses in both cultivars. Over 3000 amyloplast-localized proteins were identified, of which >98% were quantified and defined as the kfALP (kiwifruit amyloplast proteome). The kfALP data were validated by Tandem-Mass-Tag (TMT) labeled proteomics in 'Hort16A'. Analysis of the proteomic data across development and ripening revealed: 1) a conserved increase in the abundance of proteins participating in starch synthesis/degradation during both amyloplast B&D; 2) up-regulation of proteins for chlorophyll degradation and of plastoglobule-localized proteins associated with chloroplast breakdown and plastoglobule formation during amyloplast differentiation; 3) constitutive expression of proteins involved in ATP supply and protein import during amyloplast B&D. Interestingly, two different pathways of amyloplast B&D were observed in the two cultivars. In 'Hayward', significant increases in abundance of photosynthetic- and tetrapyrrole metabolism-related proteins were observed, but the opposite trend was observed in 'Hort16A'. In conclusion, analysis of the kfALP provides new insights into the potential mechanisms underlying amyloplast B&D with relevance to key fruit quality traits in contrasting kiwifruit cultivars.

The Rice Aspartyl-tRNA Synthetase YLC3 Regulates Amino Acid Homeostasis and Chloroplast Development Under Low Temperature.

Aminoacyl tRNA synthetases primarily function to attach specific amino acids to the corresponding tRNAs during protein translation. However, their roles in regulating plant growth and development still remain elusive. Here we reported a rice thermo-sensitive mutant yellow leaf chlorosis3 (ylc3) with reduced chlorophyll content, altered thylakoid structure, and substantially elevated levels of free aspartate, asparagine and glutamine in leaves under low temperature condition. Map-based cloning identified that YLC3 encodes an aspartyl-tRNA synthetase which is localized in cytosol and mitochondria. In addition, quantitative proteomics analysis revealed that both nuclear and chloroplast-encoded thylakoid proteins were significantly down-regulated in the mutant. On the other hand, proteins involved in amino acid metabolism and the process of protein synthesis were up-regulated in ylc3, particularly for key enzymes that convert aspartate to asparagine. Moreover, uncharged tRNA-Asp accumulation and phosphorylation of the translation initiation factor eIF2α was detected in the mutant, suggesting that YLC3 regulates the homeostasis of amino acid metabolism and chloroplast thylakoid development through modulation of processes during protein synthesis.

DNA and coding/non-coding RNA methylation analysis provide insights into tomato fruit ripening.

Ripening is the last, irreversible developmental stage during which fruit become palatable, thus promoting seed dispersal by frugivory. In Alisa Craig fruit, mRNAs with increasing m5C levels, such as STPK and WRKY 40, were identified as being involved in response to biotic and abiotic stresses. Furthermore, two mRNAs involved in cell wall metabolism, PG and EXP-B1, also presented increased m5C levels. In the Nr mutant, several m5C-modified mRNAs involved in fruit ripening, including those encoding WRKY and MADS-box proteins, were found. Targets of long non-coding RNAs and circular RNAs with different m5C sites were also found; these targets included 2-alkenal reductase, soluble starch synthase 1, WRKY, MADS-box, and F-box/ketch-repeat protein SKIP11. A combined analysis of changes in 5mC methylation and mRNA revealed many differentially expressed genes with differentially methylated regions encoding transcription factors and key enzymes related to ethylene biosynthesis and signal transduction; these included ERF084, EIN3, AP2/ERF, ACO5, ACS7, EIN3/4, EBF1, MADS-box, AP2/ERF, and ETR1. Taken together, our findings contribute to the global understanding of the mechanisms underlying fruit ripening, thereby providing new information for both fruit and post-harvest behavior.

Integrative Proteome and Phosphoproteome Profiling of Early Cold Response in Maize Seedlings.

Cold limits the growth and yield of maize in temperate regions, but the molecular mechanism of cold adaptation remains largely unexplored in maize. To identify early molecular events during cold shock, maize seedlings were treated under 4 °C for 30 min and 2 h, and analyzed at both the proteome and phosphoproteome levels. Over 8500 proteins and 19,300 phosphopeptides were quantified. About 660 and 620 proteins were cold responsive at protein abundance or site-specific phosphorylation levels, but only 65 proteins were shared between them. Functional enrichment analysis of cold-responsive proteins and phosphoproteins revealed that early cold response in maize is associated with photosynthesis light reaction, spliceosome, endocytosis, and defense response, consistent with similar studies in Arabidopsis. Thirty-two photosynthesis proteins were down-regulated at protein levels, and 48 spliceosome proteins were altered at site-specific phosphorylation levels. Thirty-one kinases and 33 transcriptional factors were cold responsive at protein, phosphopeptide, or site-specific phosphorylation levels. Our results showed that maize seedlings respond to cold shock rapidly, at both the proteome and phosphoproteome levels. This study provides a comprehensive landscape at the cold-responsive proteome and phosphoproteome in maize seedlings that can be a significant resource to understand how C4 plants respond to a sudden temperature drop.

Tandem Mass Tag-Based Quantitative Proteomics Reveals Implication of a Late Embryogenesis Abundant Protein (BnLEA57) in Seed Oil Accumulation in Brassica napus L.

Enhancing oil content is one of the major goals in Brassica napus breeding; however, genetic regulation of seed oil content in plants is complex and not fully elucidated. In this study, we report proteins that were differentially accumulated in immature seeds of 35 days after anthesis between two recombinant inbred lines with contrasting seed oil content, high oil content line (HOCL) and low oil content line (LOCL) using a multiplex isobaric tandem mass tags (TMT)-based quantitative proteomic approach. Over 4,600 proteins were quantified in seeds of the two lines, and 342 proteins showed differential accumulation between seeds of HOCL and LOCL. Gene Ontology enrichment analysis revealed that the differentially accumulated proteins were enriched in proteins involved in lipid biosynthesis and metabolism, photosynthesis, and nutrient reservoir activity. Western blot confirmed the increased abundance of a late embryogenesis abundant protein (BnLEA57) in HOCL seeds compared with LOCL seeds, and overexpression of either BnLEA57 gene or its homology BnLEA55 in transgenic Arabidopsis thaliana enhanced oil content in Arabidopsis seeds. Our work provides new insights into the molecular regulatory mechanism of seed oil content in B. napus.

Quantitative proteome analysis reveals changes of membrane transport proteins in Sedum plumbizincicola under cadmium stress.

Sedum plumbizincicola is an herbaceous species tolerant of excessive cadmium accumulation in above-ground tissues. The implications of membrane proteins, especially integrative membrane proteins, in Cd detoxification of plants have received attention in recent years, but a comprehensive profiling of Cd-responsive membrane proteins from Cd hyperaccumulator plants is lacking. In this study, the membrane proteins of root, stem, and leaf tissues of S. plumbizincicola seedlings treated with Cd solution for 0, 1 or 4 days were analyzed by Tandem Mass Tag (TMT) labeling-based proteome quantification (Data are available via ProteomeXchange with identifier PXD025302). Total 3353 proteins with predicted transmembrane helices were identified and quantified in at least one tissue group. 1667 proteins were defined as DAPs (differentially abundant proteins) using fold change >1.5 with p-values <0.05. The number of DAPs involved in metabolism, transport protein, and signal transduction was significantly increased after exposure to Cd, suggesting that the synthesis and decomposition of organic compounds and the transport of ions were actively involved in the Cd tolerance process. The number of up-regulated transport proteins increased significantly from 1-day exposure to 4-day exposure, from 5 to 112, 16 to 42, 18 to 44, in root, stem, and leaf, respectively. Total 352 Cd-regulated transport proteins were identified, including ABC transporters, ion transport proteins, aquaporins, proton pumps, and organic transport proteins. Heterologous expression of SpABCB28, SpMTP5, SpNRAMP5, and SpHMA2 in yeast and subcellular localization showed the Cd-specific transport activity. The results will enhance our understanding of the molecular mechanism of Cd hypertolerance and hyperaccumulation in S. plumbizincicola and will be benefit for future genetic engineering in phytoremediation.

Data-Independent Acquisition-Based Proteome and Phosphoproteome Profiling Reveals Early Protein Phosphorylation and Dephosphorylation Events in Arabidopsis Seedlings upon Cold Exposure.

Protein phosphorylation plays an important role in mediating signal transduction in cold response in plants. To better understand how plants sense and respond to the early temperature drop, we performed data-independent acquisition (DIA) method-based mass spectrometry analysis to profile the proteome and phosphoproteome of Arabidopsis seedlings upon cold stress in a time-course manner (10, 30 and 120 min of cold treatments). Our results showed the rapid and extensive changes at the phosphopeptide levels, but not at the protein abundance levels, indicating cold-mediated protein phosphorylation and dephosphorylation events. Alteration of over 1200 proteins at phosphopeptide levels were observed within 2 h of cold treatment, including over 140 kinases, over 40 transcriptional factors and over 40 E3 ligases, revealing the complexity of regulation of cold adaption. We summarized cold responsive phosphoproteins involved in phospholipid signaling, cytoskeleton reorganization, calcium signaling, and MAPK cascades. Cold-altered levels of 73 phosphopeptides (mostly novel cold-responsive) representing 62 proteins were validated by parallel reaction monitoring (PRM). In summary, this study furthers our understanding of the molecular mechanisms of cold adaption in plants and strongly supports that DIA coupled with PRM are valuable tools in uncovering early signaling events in plants.

Improved Integrated Whole Proteomic and Phosphoproteomic Profiles of Severe Acute Pancreatitis.

Severe acute pancreatitis (SAP) is caused by complicated biological factors, and revealing its complex pathogenesis by single-target analysis is difficult. Systematic studies have developed slowly because extraction of degradable pancreatic proteins exposed to multiple proteases is challenging. We present integrated whole proteomic and phosphoproteomic profiles of SAP rats based on a modified protein extraction strategy with less protein degradation. Data-dependent acquisition (DDA) and data-independent acquisition (DIA) strategies were applied to select an appropriate method. Total 275 differentially expressed proteins and 757 differentially expressed phosphorylated proteins were identified by DIA-based quantitative proteomics. Several signal transduction pathways, including the AMPK, MAPK, and PI3K-Akt pathways, were enriched in SAP. Up-regulation of phosphorylated proteins involved in the process of TNFA signaling and inflammatory response was also detected in SAP. Our results improve the understanding of SAP development and progression.

Identification and Functional Characterization of Tomato CircRNAs Derived from Genes Involved in Fruit Pigment Accumulation.

CircRNAs, a class of widespread circular RNAs produced from precursor mRNA back-splicing, have been implicated in regulation of gene expression in eukaryotes, but their biological functions in plants have not yet been elucidated. By deep sequencing of rRNA-removed and RNase R-digested RNA samples we have identified several thousands of putative back-splicing sites in tomato fruit (Solanum lycopersicum) and show that the abundance of some of these circRNAs derived from fruit pigment biosynthesis genes are regulated by fruit ripening. Herein, we overexpressed a circRNA derived from Phytoene Synthase 1 (PSY1) in tomato 'Ailsa Craig' and microTom. The PSY1 mRNA abundance, the lycopene and ß-carotene accumulation were decreased significantly in the transgenic tomato fruits, likely due to the continuous highly expressed circRNAs and/or the low abundant linear RNAs generated from the overexpression vector. Besides, overexpression of a circRNA derived from Phytoene Desaturase (PDS) showed similar results. Our results provide biological insights into plant circRNAs.

Isolation and characterization of a novel cadmium-regulated Yellow Stripe-Like transporter (SnYSL3) in Solanum nigrum.

KEY MESSAGE: SnYSL3 encodes a plasma-localized transporter delivering various metal-nicotianamine complexes. The expression of SnYSL3 is up-regulated by excess Cd, suggesting an important role for SnYSL3 in response to Cd stress. The Yellow Stripe-Like (YSL) transporters have been proposed to participate in metal uptake and long-range transport in model plants. In this study, we isolated and characterized a novel member of the YSL gene family, SnYSL3, from the cadmium hyperaccumulator Solanum nigrum. SnYSL3 was constitutively expressed and encodes a plasma membrane-localized protein. In situ RNA hybridization localized the SnYSL3 transcripts predominantly in vascular tissues and epidermal cells of the roots and stems, while in leaves, the mRNA levels were high in the vasculature. The SnYSL3 expression level was up-regulated by excess Cd, excess Fe and Cu deficiency. Heterologous expression of SnYSL3 in yeast revealed that SnYSL3 transports nicotianamine complexes containing Fe(II), Cu, Zn and Cd. SnYSL3 overexpression in Arabidopsis thaliana decreased Fe and Mn concentrations in the roots and increased the root-to-shoot translocation ratios of Fe and Mn. Under Cd exposure, the transgenic plants showed increased translocation ratios of Fe and Cd, but no difference was observed in Mn translocation from roots to shoots between the transgenic and wild-type lines. Although the accurate function of SnYSL3 remains to be confirmed, these results suggest that SnYSL3 is a transporter delivering a broad range of metal-nicotianamine complexes and is potentially important for the response to heavy metal stress, especially due to Cd and Fe.

The N-terminal degenerated metal-binding domain is involved in the heavy metal transport activity of TaHMA2.

KEY MESSAGE: We identified key residues of TaHMA2, and the N- and C-terminal regions of the protein have different roles in its transport function when heterologously expressed in yeast. TaHMA2, a P1B-type ATPase from wheat (Triticum aestivum L.), plays an important role in heavy metal homeostasis in plants. A previous study showed that overexpressing TaHMA2 in rice (Oryza sativa L.), Arabidopsis thaliana, or tobacco (Nicotiana tabacum L.) resulted in various responses to heavy metals. Here, we report the heterologous expression of TaHMA2 in the yeast Saccharomyces cerevisiae. TaHMA2 expression increased the yeast's sensitivity to Cd, but not to Zn, Pb or Co, and increased Cd accumulation was concurrently observed. The eGFP-TaHMA2 fusion protein was localized to the plasma membrane and showed a discontinuous pattern. Mutagenesis of the cysteine and glutamate residues in the N-terminal metal-binding domain (N-MBD) impaired the function of TaHMA2. Deletion of most of the C terminus (TaHMA2ΔC, 712-1003) partially abolished the protein's function, whereas deletion of the N terminus (TaHMA2ΔN, 2-699) completely abolished Cd sensitivity. These data suggest that cysteine and glutamate residues are important for the metal-binding/translocation function of TaHMA2. Additional studies are needed to further understand the selectivity of TaHMA2 in planta.

Requirement of CHROMOMETHYLASE3 for somatic inheritance of the spontaneous tomato epimutation Colourless non-ripening.

Naturally-occurring epimutants are rare and have mainly been described in plants. However how these mutants maintain their epigenetic marks and how they are inherited remain unknown. Here we report that CHROMOMETHYLASE3 (SlCMT3) and other methyltransferases are required for maintenance of a spontaneous epimutation and its cognate Colourless non-ripening (Cnr) phenotype in tomato. We screened a series of DNA methylation-related genes that could rescue the hypermethylated Cnr mutant. Silencing of the developmentally-regulated SlCMT3 gene results in increased expression of LeSPL-CNR, the gene encodes the SBP-box transcription factor residing at the Cnr locus and triggers Cnr fruits to ripen normally. Expression of other key ripening-genes was also up-regulated. Targeted and whole-genome bisulfite sequencing showed that the induced ripening of Cnr fruits is associated with reduction of methylation at CHG sites in a 286-bp region of the LeSPL-CNR promoter, and a decrease of DNA methylation in differentially-methylated regions associated with the LeMADS-RIN binding sites. Our results indicate that there is likely a concerted effect of different methyltransferases at the Cnr locus and the plant-specific SlCMT3 is essential for sustaining Cnr epi-allele. Maintenance of DNA methylation dynamics is critical for the somatic stability of Cnr epimutation and for the inheritance of tomato non-ripening phenotype.

Manganese-mitigation of cadmium toxicity to seedling growth of Phytolacca acinosa Roxb. is controlled by the manganese/cadmium molar ratio under hydroponic conditions.

Manganese (Mn) can interact with cadmium (Cd) in environments and influence the toxic effect of Cd on plants. However, few studies have investigated the relationship between the Mn/Cd ratio and plant Cd-toxicity along Cd concentrations. In this paper, we studied the effects of external Mn/Cd molar ratios (0, 10, 30, 50 and 60) on Cd toxicity in the Mn hyperaccumulator and Cd tolerant plant, Phytolacca acinosa Roxb., at three Cd levels (50, 100 and 200 µM) under hydroponic conditions. Our result showed that seedling growth (y) under Cd stress was strongly positively related to the solution Mn/Cd molar ratio (SMCR). The relationship between the two variables under solution Cd concentrations was well explained by the linear regression model y=a+b1 (SMCR)+b2 (Solution-Cd). Increasing SMCR significantly reduced the Cd concentration and increased the Mn concentration in plant tissues. However, seedling growth was consistent with the shoot Mn/Cd molar ratio rather than with the Mn or Cd concentrations in plant tissues. At low levels of SMCR (e.g. 0 and 10), elevation of Mn distribution in shoot tissues might be a mechanism in P. acinosa seedlings to defend against Cd-toxicity. In comparison with low levels of SMCR, high levels of SMCR (e.g. 50 and 60) greatly alleviated lipid peroxidation and plant water-loss, and enhanced photosynthesis. However, the alleviated lipid peroxidation in the Mn-mitigation of Cd toxicity was likely to be the secondary effect resulting from the antagonism between Mn and Cd in the plant.

Functional analyses of TaHMA2, a P(1B)-type ATPase in wheat.

Currently, there are few studies concerning the function of heavy metal ATPase 2 (HMA2), particularly in monocotyledons, and the potential application of this protein in biofortification and phytoremediation. Thus, we isolated and characterized the TaHMA2 gene from wheat (Triticum aestivum L.). Our results indicate that TaHMA2 is localized to the plasma membrane and stably expressed, except in the nodes, which showed relatively high expression. Zinc/cadmium (Zn/Cd) resistance was observed in TaHMA2-transformed yeast. The over-expression of TaHMA2 increased the elongation and decreased the seed-setting rate in rice (Oryza sativa L.), but not Arabidopsis thaliana, tobacco (Nicotiana tabacum L.) or wheat. TaHMA2 over-expression also improved root-shoot Zn/Cd translocation, especially in rice. The seeds of transgenic rice and wheat, not tobacco, showed decreased Zn concentrations. The Zn concentration was decreased in all parts of the transgenic rice seeds, but was decreased only in the ventral endosperm of wheat, which showed an increased Zn concentration in the embryo and aleurone. The over-expression of TaHMA2 improved plant tolerance under moderate Zn stress and Zn deficiency, but Zn and Cd resistance decreased under high levels of Zn and Cd stress, respectively. The Cd concentration in transgenic rice seedlings was dramatically increased under Zn deficiency. Thus, over-expression of TaHMA2 showed a more obvious phenotype in monocotyledons than in dicotyledons. These findings provide important information for TaHMA2, and more efforts should be made in the future to characterize the reduced Zn concentration in TaHMA2 transgenic grains and the diversity of TaHMA2 substrate specificity.

The effects of copper, manganese and zinc on plant growth and elemental accumulation in the manganese-hyperaccumulator Phytolacca americana.

Synchrotron radiation X-ray fluorescence (SRXRF) and inductively coupled plasma mass spectrometry were used to estimate major, minor and trace elements in Cu-, Zn- and Mn-treated Phytolacca americana. The effects of the addition of Cu, Zn and Mn on morphological parameters, such as root length, shoot height, and fresh and dry weights of shoots and roots, were also examined. In addition, the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidases (GPX) and catalase (CAT) and the expression of Fe-SOD, Cu/Zn-SOD, metallothionein-2 and glutathione S-transferase (GST) exposed to the highest amounts of Cu, Zn or Mn were detected. Our results confirmed the following: (1) Zn supplementation leads to chlorosis, disturbed elemental homeostasis and decreased concentrations of micro- and macroelements such as Fe, Mg, Mn, Ca and K. Cu competed with Fe, Mn and Zn uptake in plants supplemented with 25 µM Cu. However, no antagonistic interactions took place between Cu, Zn, Mn and Fe uptake in plants supplemented with 100 µM Cu. Mn supplementation at various concentrations had no negative effects on elemental deficits. Mn was co-located with high concentrations of Fe and Zn in mature leaves and the concentrations of macro elements were unchanged. (2) P. americana supplemented with increased concentrations of Zn and Cu exhibited lower biomass production and reduced plant growth. (3) When plants were supplemented with the highest Zn and Cu concentrations, symptoms of toxicity corresponded to decreased SOD or CAT activities and increased APX and GPX activities. However, Mn tolerance corresponded to increased SOD and CAT activities and decreased POD and APX activities. Our study revealed that heavy metals partially exert toxicity by disturbing the nutrient balance and modifying enzyme activities that induce damage in plants. However, P. americana has evolved hyper accumulating mechanisms to maintain elemental balance and redox homeostasis under excess Mn.