The NAC transcription factor MdNAC4 positively regulates nitrogen deficiency-induced leaf senescence by enhancing ABA biosynthesis in apple.

Although it is well established that nitrogen (N) deficiency induces leaf senescence, the molecular mechanism of N deficiency-induced leaf senescence remains largely unknown. Here, we show that an abscisic acid (ABA)-responsive NAC transcription factor (TF) is involved in N deficiency-induced leaf senescence. The overexpression of MdNAC4 led to increased ABA levels in apple calli by directly activating the transcription of the ABA biosynthesis gene MdNCED2. In addition, MdNAC4 overexpression promoted N deficiency-induced leaf senescence. Further investigation showed that MdNAC4 directly bound the promoter of the senescence-associated gene (SAG) MdSAG39 and upregulated its expression. Interestingly, the function of MdNAC4 in promoting N deficiency-induced leaf senescence was enhanced in the presence of ABA. Furthermore, we identified an interaction between the ABA receptor protein MdPYL4 and the MdNAC4 protein. Moreover, MdPYL4 showed a function similar to that of MdNAC4 in ABA-mediated N deficiency-induced leaf senescence. These findings suggest that ABA plays a central role in N deficiency-induced leaf senescence and that MdPYL4 interacts with MdNAC4 to enhance the response of the latter to N deficiency, thus promoting N deficiency-induced leaf senescence. In conclusion, our results provide new insight into how MdNAC4 regulates N deficiency-induced leaf senescence.

The NAC transcription factor MdNAC29 negatively regulates drought tolerance in apple.

Drought stress is an adverse stimulus that affects agricultural production worldwide. NAC transcription factors are involved in plant development and growth but also play different roles in the abiotic stress response. Here, we isolated the apple MdNAC29 gene and investigated its role in regulating drought tolerance. Subcellular localization experiments showed that MdNAC29 was localized to the nucleus and transcription was induced by the PEG treatment. Over-expression of MdNAC29 reduced drought tolerance in apple plants, calli, and tobacco, and exhibited higher relative conductivity, malondialdehyde (MDA) content, and lower chlorophyll content under drought stress. The transcriptomic analyses revealed that MdNAC29 reduced drought resistance by modulating the expression of photosynthesis and leaf senescence-related genes. The qRT-PCR results showed that overexpression of MdNAC29 repressed the expression of drought-resistance genes. Yeast one-hybrid and dual-luciferase assays demonstrated that MdNAC29 directly repressed MdDREB2A expression. Moreover, the yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that MdNAC29 interacted with the MdPP2-B10 (F-box protein), which responded to drought stress, and MdPP2-B10 enhanced the repressive effect of MdNAC29 on the transcriptional activity of the MdDREB2A. Taken together, our results indicate that MdNAC29 is a negative regulator of drought resistance, and provide a theoretical basis for further molecular mechanism research.

BFP Net: Balanced Feature Pyramid Network for Small Apple Detection in Complex Orchard Environment.

Despite of significant achievements made in the detection of target fruits, small fruit detection remains a great challenge, especially for immature small green fruits with a few pixels. The closeness of color between the fruit skin and the background greatly increases the difficulty of locating small target fruits in the natural orchard environment. In this paper, we propose a balanced feature pyramid network (BFP Net) for small apple detection. This network can balance information mapped to small apples from two perspectives: multiple-scale fruits on the different layers of FPN and a characteristic of a new extended feature from the output of ResNet50 conv1. Specifically, we design a weight-like feature fusion architecture on the lateral connection and top-down structure to alleviate the small-scale information imbalance on the different layers of FPN. Moreover, a new extended layer from ResNet50 conv1 is embedded into the lowest layer of standard FPN, and a decoupled-aggregated module is devised on this new extended layer of FPN to complement spatial location information and relieve the problem of locating small apple. In addition, a feature Kullback-Leibler distillation loss is introduced to transfer favorable knowledge from the teacher model to the student model. Experimental results show that APS of our method reaches 47.0%, 42.2%, and 35.6% on the benchmark of the GreenApple, MinneApple, and Pascal VOC, respectively. Overall, our method is not only slightly better than some state-of-the-art methods but also has a good generalization performance.

Transcriptome analysis reveals candidate genes involved in nitrogen deficiency stress in apples.

Nitrogen is one of the macroelements required for plant growth and development and the identification of candidate genes involved in nitrogen deficiency stress is of great importance to the sustainable development of agriculture. Here, we found that the color of apple leaves changed from dark green to yellow-green, the malondialdehyde (MDA) content, soluble protein content, and proline content significantly increased, the chlorophyll content significantly decreased in response to nitrate deficiency stress. According to the physiological and biochemical changes of apple leaves during nitrate deficiency stress, nitrogen deficiency stress was divided into two stages: early nitrogen deficiency stage (ES) and late nitrogen deficiency stage (LS). Transcriptome sequencing was performed in these two stress stages. 5773 differential expression genes (DEGs) were identified in the early nitrogen deficiency stress stage and 6130 DEGs were identified in the late nitrogen deficiency stress stage. Functional analysis of these DEGs revealed that a large number of DEGs were enriched in 'porphyrin and chlorophyll metabolic' pathways, the 'photosynthesis' pathway, the 'photosynthesis-antenna protein' pathway, and the 'ABA', 'ETH', and 'JA' signal transduction pathways, and the metabolic networks of these pathways were constructed. In addition, overexpression of MdNAC4 weakened the tolerance of apple calli to nitrogen deficiency stress. Taken together, our results reveal possible pathways for apple adaptation to nitrogen deficiency stress and identify the function of MdNAC4, a key transcription factor regulating nitrogen deficiency stress, which enriches the molecular mechanism of apple adapting to a nitrogen deficiency environment.

The apple GARP family gene MdHHO3 regulates the nitrate response and leaf senescence.

The regulation of plant gene expression by nitrate is a complex regulatory process. Here, we identified 90 GARP family genes in apples by genome-wide analysis. As a member of the GARP gene family, the expression of MdHHO3 (Malus domestica HYPERSENSITIVITY TO LOW PHOSPHATE-ELICITED PRIMARY ROOT SHORTENING1 HOMOLOG 3) is upregulated under N (nitrogen) supply. The results of DNA-binding site analysis and electrophoretic mobility shift assays (EMSA) showed that MdHHO3 binds to the motif-containing GAATC. Furthermore, MdHHO3 binds to its promoter sequence and inhibits its activity. In addition, the overexpression of MdHHO3 in apple calli resulted in less accumulation of nitrate in 35S:MdHHO3-GFP calli and downregulated the expression of the nitrate transport-related genes but upregulated the expression of the nitrate assimilation-related genes. Similarly, the expression of the nitrate transport-related genes was downregulated and the expression of the nitrate assimilation-related genes was upregulated in MdHHO3 overexpression Arabidopsis and tobacco plants. Interaction experiments showed that MdHHO3 could bind to the promoter MdNRT2.1 (NITRATE TRANSPORTER 2.1) and negatively regulate its expression. Moreover, the exposure of MdHHO3-overexpressing Arabidopsis and tobacco to nitrate deficiency resulted in an early senescence phenotype as compared to the WT plants. These results show that MdHHO3 can not only negatively regulate nitrate accumulation in response to nitrate but also promote early leaf senescence under nitrate deficiency. This information may be useful to further reveal the mechanism of the nitrate response and demonstrates that nitrate deficiency induces leaf senescence in apples.

PpMYB52 negatively regulates peach bud break through the gibberellin pathway and through interactions with PpMIEL1.

Bud dormancy, which enables damage from cold temperatures to be avoided during winter and early spring, is an important adaptive mechanism of deciduous fruit trees to cope with seasonal environmental changes and temperate climates. Understanding the regulatory mechanism of bud break in fruit trees is highly important for the artificial control of bud break and the prevention of spring frost damage. However, the molecular mechanism underlying the involvement of MYB TFs during the bud break of peach is still unclear. In this study, we isolated and identified the PpMYB52 (Prupe.5G240000.1) gene from peach; this gene is downregulated in the process of bud break, upregulated in response to ABA and downregulated in response to GA. Overexpression of PpMYB52 suppresses the germination of transgenic tomato seeds. In addition, Y2H, Bimolecular fluorescence complementation (BiFC) assays verified that PpMYB52 interacts with a RING-type E3 ubiquitin ligase, PpMIEL1, which is upregulated during bud break may positively regulate peach bud break by ubiquitination-mediated degradation of PpMYB52. Our findings are the first to characterize the molecular mechanisms underlying the involvement of MYB TFs in peach bud break, increasing awareness of dormancy-related molecules to avoid bud damage in perennial deciduous fruit trees.

MdNAC4 Interacts With MdAPRR2 to Regulate Nitrogen Deficiency-Induced Leaf Senescence in Apple (Malus domestica).

Nitrogen (N) is one of the important macronutrients in plants, and N deficiency induces leaf senescence. However, the molecular mechanism underlying how N deficiency affects leaf senescence is unclear. Here, we report an apple NAC TF, MdNAC4, that participates in N deficiency-induced leaf senescence. The senescence phenotype of apple leaves overexpressing MdNAC4 was enhanced after N deficiency. Consistently, the chlorophyll content of transgenic leaves was significantly lower than that in the WT control leaves, the expression of chlorophyll catabolism-related genes (MdNYC1, MdPAO, and MdSGR1) was significantly higher than that in the WT controls, and the expression of chlorophyll synthesis-related genes (MdHEMA, MdCHLI, and MdCHLM) was significantly lower than that in the WT control leaves. Furthermore, MdNAC4 was found to directly activate the transcription of the chlorophyll catabolism-related genes MdNYC1 and MdPAO. Additionally, MdNAC4 was proven to interact with MdAPRR2 proteins both in vitro and in vivo, and overexpression of MdAPRR2 seemed to delay N deficiency-induced leaf senescence. Correspondingly, the chlorophyll loss of MdAPRR2-overexpressing (MdAPRR2-OE) lines was significantly lower than in WT control plants. Although downregulated, the expression of the chlorophyll synthesis-related genes MdHEMA, MdCHLI, and MdCHLM in the transgenic plants was more than twice that in the WT control plants. Taken together, our results enrich the regulatory network of leaf senescence induced by N deficiency through the interaction between MdNAC4 and MdAPRR2.

Prunus persica transcription factor PpNAC56 enhances heat resistance in transgenic tomatoes.

Members of the NAC (NAM, ATAF1,2 and CUC2) transcription factor family are involved in numerous processes of plant growth and development and play an important role in the response to abiotic stresses such as salinity, drought and heat, but little research on this topic has been done in peach. In this study, we analyzed the expression patterns of PpNAC56 under abiotic stress and found that PpNAC56 responded to high-temperature stress. To verify the function of PpNAC56, we overexpressed this gene in tomato plants and found that, compared with WT plants, the transgenic tomato plants could accumulate more osmoregulatory substances after high-temperature treatment and thus were more heat resistance. Then, using Y2H, BIFC, and pull-down assays, we found that PpNAC56 could interact with PpMIEL1. In addition, Y1H and dual-luciferase assays verified that PpNAC56 could activate the expression of PpHSP17.4 and PpSnRK2D. The above experimental results demonstrate that PpNAC56 plays an important role in the plant response to heat stress.

Prunus persica Terpene Synthase PpTPS1 Interacts with PpABI5 to Enhance Salt Resistance in Transgenic Tomatoes.

Terpene synthase (TPS) is related to the production of aromatic substances, but there are few studies on the impact of abiotic stress on TPS and its molecular mechanism, especially in peaches. This study found that salt resistance and abscisic acid (ABA) sensitivity of transgenic tomatoes were enhanced by overexpression of PpTPS1. Moreover, it was found that PpTPS1 interacted with and antagonized the expression of the bZIP transcription factor ABA INSENSITIVE 5 (PpABI5), which is thought to play an important role in salt suitability. In addition, PpTCP1, PpTCP13, and PpTCP15 were found to activate the expression of PpTPS1 by yeast one-hybrid (Y1H) and dual-luciferase assays, and they could also be induced by ABA. In summary, PpTPS1 may be involved in the ABA signaling regulatory pathway and play an important role in salt acclimation, providing a new reference gene for the improvement of salt resistance in peaches.

Genome-wide identification and characterization of the Prunus persica ferredoxin gene family and its role in improving heat tolerance.

Ferredoxin is involved in many biological processes, such as carbon fixation, nitrogen assimilation, chlorophyll metabolism, and fatty acid synthesis, and it plays a role in plant resistance to stress. However, the functions of Fds in peach during stress are unclear. In this study, 11 members of the peach Fd gene family were identified and divided into six groups (I- VI). We carried out bioinformatics analysis on these sequences, analyzed the physical and chemical properties of PpFd protein and the cis-elements in its promoter region, and predicted and compared the differences in gene structure and conserved protein motifs among groups. The results showed that the PpFd protein was highly conserved in plant species. In addition, overexpression of PpFd08 significantly increased the tolerance of transgenic tomato to high-temperature stress. The transcriptome analysis and qRT-PCR results of PpFd08 transgenic apple calli showed that PpFd08 might enhance heat resistance by modulating the expression of heat tolerance related genes. The results of this study provide a new understanding for the further study of the function of PpFd protein in peach and a candidate gene for improving the heat resistance of peach.

China's agricultural GHG emission efficiency: regional disparity and spatial dynamic evolution.

Improving China's agricultural greenhouse gases (GHG) emission efficiency has become an important way to cope with climate change in an ecologically-and ethically responsible manner. In this paper, we use a global slacks-based inefficiency model to evaluate the agricultural greenhouse gases (GHG) emission efficiency levels in China during 2000-2015. The regional disparity of China's GHG emission efficiency is examined by using a Dagum Gini coefficient. A spatial Markov chain technique is also employed to investigate the spatial dynamic evolution of agricultural GHG emission efficiency. The results show that: (1) China's agricultural GHG emission efficiency increased steadily during the study period; a certain gap in efficiency among provinces and regions also exists. (2) Between-group disparity is the main source of the overall regional disparities in China's agricultural GHG emission efficiency. The disparities between regions are on the rise, while the disparities within regions are relatively stable. (3) China's agricultural GHG emission efficiency demonstrates significant spatial dependence. This study provides policy implications for the sustainable development of China's agricultural sector.

Does the promotion pressure on local officials matter for regional carbon emissions? Evidence based on provincial-level leaders in China.

This paper constructs data from 30 provinces in mainland China from 1997 to 2016 and mainly adopts panel data fixed effects models to investigate how the promotion pressure on local officials affects regional carbon emissions. Our empirical results show that the relationship between the promotion pressure on local officials and regional carbon emissions has a dynamic evolution characteristic during our research period. Specifically, the promotion pressure on local officials is positively associated with regional carbon emissions before 2009; however, this relationship weakened after China's carbon emission regulatory policies were strengthened in 2010. Furthermore, our heterogeneity analysis results show that the effect of promotion pressure on regional carbon emissions is moderated by the regional industrial structure, the economic development level, regional innovation capability, the tenure of officials and the age of officials. The conclusions of this study are helpful for understanding the driving factors of regional carbon emissions from the political economy perspective, and they also have implications for the formulation of performance evaluation and carbon emission reduction policies.

How does anti-corruption affect enterprise green innovation in China's energy-intensive industries?

Rampant corruption exists in China's energy-intensive industries. However, we know little about the nexus of corruption and enterprise green innovation in China's energy-intensive industries. This paper discusses the impact of anti-corruption on enterprises' green innovation and its effect margin. Analyzing the panel data of Chinese listed enterprises in energy-intensive industries from 2009 to 2017, we find that anti-corruption played a positive role in stimulating enterprises' green innovation investments in energy-intensive industries. Then we adopt the instrumental variable approach and difference-in-differences model to alleviate the endogeneity problem. Moreover, we find that research and development investments from state-owned, high-tech enterprises and enterprises in the regions with more government intervention or weaker intellectual property protection were more prominent after the anti-corruption campaign. Finally, political connection played an intermediary role in this process, in which only the government-official political connection worked. Our results highlight the roles of enterprises' attributes and environmental characteristics as important factors in the relationship between anti-corruption and green innovation investments. Policymakers should enhance the control of corruption to boost green innovation in energy-intensive industries.

OVATE Family Protein PpOFP1 Physically Interacts With PpZFHD1 and Confers Salt Tolerance to Tomato and Yeast.

The OVATE family protein (OFP) genes (OFPs) have been shown to respond to salt stress in plants. However, the regulatory mechanism for salt tolerance of the peach (Prunus persica) OFP gene PpOFP1 has not been elucidated. In this study, using yeast two-hybrid screening, we isolated a nucleus-localized ZF-HD_dimer domain protein PpZFHD1, which interacts with the PpOFP1 protein in the peach cultivar "Zhongnongpan No.10". A segmentation experiment further suggested that the interaction happens more specifically between the N-terminal, contains ZF-HD_dimer domain, of PpZFHD1 and the C-terminal, consists of OVATE domain, of PpOFP1. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) experiments indicate that transcription of these two genes are induced by 200 mmol/L (mM) NaCl treatment. Heterogeneous transformation experiments suggested that the growth status of transformed yeast strain over-expressing each of these two genes was more robust than that of control (CK). Furthermore, transgenic tomato plants over-expressing PpOFP1 were also more robust. They had a higher content of chlorophyll, soluble proteins, soluble sugars, and proline. Activities of the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in these plants were higher, and tissues from these plants exhibited a lower relative conductivity and malondialdehyde (MDA) content. These results suggest that PpOFP1 physically interacts with PpZFHD1 and confers salt tolerance to tomato and yeast, thus revealing a novel mechanism for regulating salt tolerance in peach and other perennial deciduous trees.

Chromosome-level genome assemblies of five Prunus species and genome-wide association studies for key agronomic traits in peach.

Prunus species include many important perennial fruit crops, such as peach, plum, apricot, and related wild species. Here, we report de novo genome assemblies for five species, including the cultivated species peach (Prunus persica), plum (Prunus salicina), and apricot (Prunus armeniaca), and the wild peach species Tibetan peach (Prunus mira) and Chinese wild peach (Prunus davidiana). The genomes ranged from 240 to 276 Mb in size, with contig N50 values of 2.27-8.30 Mb and 25,333-27,826 protein-coding gene models. As the phylogenetic tree shows, plum diverged from its common ancestor with peach, wild peach species, and apricot ~7 million years ago (MYA). We analyzed whole-genome resequencing data of 417 peach accessions, called 3,749,618 high-quality SNPs, 577,154 small indels, 31,800 deletions, duplications, and inversions, and 32,338 insertions, and performed a structural variant-based genome-wide association study (GWAS) of key agricultural traits. From our GWAS data, we identified a locus associated with a fruit shape corresponding to the OVATE transcription factor, where a large inversion event correlates with higher OVATE expression in flat-shaped accessions. Furthermore, a GWAS revealed a NAC transcription factor associated with fruit developmental timing that is linked to a tandem repeat variant and elevated NAC expression in early-ripening accessions. We also identified a locus encoding microRNA172d, where insertion of a transposable element into its promoter was found in double-flower accessions. Thus, our efforts have suggested roles for OVATE, a NAC transcription factor, and microRNA172d in fruit shape, fruit development period, and floral morphology, respectively, that can be connected to traits in other crops, thereby demonstrating the importance of parallel evolution in the diversification of several commercially important domesticated species. In general, these genomic resources will facilitate functional genomics, evolutionary research, and agronomic improvement of these five and other Prunus species. We believe that structural variant-based GWASs can also be used in other plants, animal species, and humans and be combined with deep sequencing GWASs to precisely identify candidate genes and genetic architecture components.

Endodormancy Release Can Be Modulated by the GA4-GID1c-DELLA2 Module in Peach Leaf Buds.

Gibberellin (GA) plays a key role in the release of bud dormancy and the GA receptor GID1 (GIBBERELLIN INSENSITIVE DWARF1) and DELLA protein are the GA signaling parts, but the molecular mechanism of GA-GID1-DELLA module regulating leaf bud dormancy in peach (Prunus persica) is still not very clear. In this study, we isolated and characterized the GID1 gene PpGID1c from the peach cultivar "Zhong you No.4." Overexpressing PpGID1c in Arabidopsis promoted seed germination, which indicated that PpGID1c has an important function in dormancy. The expression level of PpGID1c in peach leaf buds during endodormancy release was higher than that during ecodormancy and was positively correlated with GA4 levels. Our study also found that GA4 had the most obvious effect on promoting the bud break, indicating that GA4 may be the key gibberellin to promoting peach leaf bud endodormancy release. Moreover, a quantitative real-time PCR (qRT-PCR) found that GA4 could increase the expression of the gibberellin signaling gene PpDELLA2. A yeast two-hybrid (Y2H) assay suggested that the PpGID1c interaction with the PpDELLA1 protein was not dependent on gibberellin, while the PpGID1c interaction with PpDELLA2 required GA4 or another gibberellin. These findings suggested that the GA4-GID1c-DELLA2 module regulates peach leaf bud endodormancy release, with this finding significantly enhancing our comprehensive understanding of bud endodormancy release and revealing a new mechanism for regulating leaf bud endodormancy release in peach.

Overexpression of the Peach Transcription Factor Early Bud-Break 1 Leads to More Branches in Poplar.

Shoot branching is an important adaptive trait that determines plant architecture. In a previous study, the Early bud-break 1 (EBB1) gene in peach (Prunus persica var. nectarina) cultivar Zhongyou 4 was transformed into poplar (Populus trichocarpa). PpEBB1-oe poplar showed a more branched phenotype. To understand the potential mechanisms underlying the EBB1-mediated branching, transcriptomic and proteomics analyses were used. The results showed that a large number of differentially expressed genes (DEGs)/differentially expressed proteins (DEPs) associated with light response, sugars, brassinosteroids (BR), and nitrogen metabolism were significantly enriched in PpEBB1-oe poplar. In addition, contents of sugars, BR, and amino acids were measured. Results showed that PpEBB1 significantly promoted the accumulation of fructose, glucose, sucrose, trehalose, and starch. Contents of brassinolide (BL), castasterone (CS), and 6-deoxocathasterone (6-deoxoCS) were all significantly changed with overexpressing PpEBB1. Various types of amino acids were measured and four of them were significantly improved in PpEBB1-oe poplar, including aspartic acid (Asp), arginine (Arg), cysteine (Cys), and tryptohpan (Trp). Taken together, shoot branching is a process controlled by a complex regulatory network, and PpEBB1 may play important roles in this process through the coordinating multiple metabolic pathways involved in shoot branching, including light response, phytohormones, sugars, and nitrogen.

BTB-TAZ Domain Protein PpBT3 modulates peach bud endodormancy by interacting with PpDAM5.

The dormancy-associated MADS-box (DAM) gene DAM5 has crucial roles in bud endodormancy; however, the molecular regulatory mechanism of PpDAM5 in peach (Prunus persica) has not been elucidated. In this study, using yeast two-hybrid screening, we isolated a BTB-TAZ Domain Protein PpBT3, which interacts with PpDAM5 protein, in the peach cultivar 'Chun xue'. As expected, we found that abscisic acid (ABA) maintained bud endodormancy and induced expression of the PpDAM5 gene, and that over-expressing PpDAM5 in Arabidopsis thaliana repressed seed germination. In contrast, over-expressing PpBT3 in A. thaliana promoted seed germination, and conferred resistance to ABA-mediated germination inhibition. Additionally, a qRT-PCR (quantitative real-time polymerase chain reaction) experiment suggested that the transcript level of PpBT3 gradually increased towards the endodormancy release period, which is the opposite trend of the expression pattern of PpDAM5. Our results suggest that PpBT3 modulates peach bud endodormancy by interacting with PpDAM5, thus revealing a new mechanism for regulating bud dormancy of perennial deciduous trees.

Auxin alters sodium ion accumulation and nutrient accumulation by playing protective role in salinity challenged strawberry.

High salinity in soil affects the strawberry production and fruit quality. Auxin-primed plants have enhanced responses to soil salinization. In this study, we report that exogenous application of IAA can partially relieve stress responses of strawberry seedlings. Cytological analysis showed that the ultrastructure of root tip and leaf cells in strawberry seedlings were altered under high salinity condition, which was partially recovered after the application of IAA. The study showed that the ultrastructure of root tip and leaf cells in strawberry seedlings were altered under salt stress condition, which was partially recovered after the application of IAA. Exogenous IAA ameliorated deleterious effects on seedling growth under salinity were attributed to accelerated Na+ fluxes, decreased the contents of Na+ to maintain the ion homeostasis, protect root growth, and promote the absorption of nutrients for improved photosynthetic efficiency in strawberry.