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Background Leaf colour mutations are universally expressed at the seedling stage and are ideal materials for exploring the chlorophyll biosynthesis pathway, carotenoid metabolism and the flavonoid biosynthesis pathway in plants. Results In this research, we analysed the different degrees of albinism in apple ( Malus domestica ) seedlings, including white-leaf mutants (WM), piebald leaf mutants (PM), light-green leaf mutants (LM) and normal leaves (NL) using bisulfite sequencing (BS-seq) and RNA sequencing (RNA-seq). There were 61,755, 79,824, and 74,899 differentially methylated regions (DMRs) and 7566, 3660, and 3546 differentially expressed genes (DEGs) identified in the WM/NL, PM/NL and LM/NL comparisons, respectively. Conclusion The analysis of the methylome and transcriptome showed that 9 DMR-associated DEGs were involved in the carotenoid metabolism and flavonoid biosynthesis pathway. The expression of different transcription factors (TFs) may also influence the chlorophyll biosynthesis pathway, carotenoid metabolism and the flavonoid biosynthesis pathway in apple leaf mutants. This study provides a new method for understanding the differences in the formation of apple seedlings with different degrees of albinism.

Nitrogen (N) and phosphorus (Pi) are essential macronutrients that affect plant growth and development by influencing the molecular, metabolic, biochemical, and physiological responses at the local and whole levels in plants. N and Pi stresses suppress the physiological activities of plants, resulting in agricultural productivity losses and severely threatening food security. Accordingly, plants have elaborated diverse strategies to cope with N and Pi stresses through maintaining N and Pi homeostasis. MicroRNAs (miRNAs) as potent regulators fine-tune N and Pi signaling transduction that are distinct and indivisible from each other. Specific signals, such as noncoding RNAs (ncRNAs), interact with miRNAs and add to the complexity of regulation. Elucidation of the mechanisms by which miRNAs regulate N and Pi signaling transduction aids in the breeding of plants with strong tolerance to N and Pi stresses and high N and Pi use efficiency by fine-tuning MIR genes or miRNAs. However, to date, there has been no detailed and systematic introduction and comparison of the functions of miRNAs in N and Pi signaling transduction from the perspective of miRNAs and their applications. Here, we summarized and discussed current advances in the involvement of miRNAs in N and Pi signaling transduction and highlighted that fine-tuning the MIR genes or miRNAs involved in maintaining N and Pi homeostasis might provide valuable sights for sustainable agriculture.

In apple (Malus) cultivation, the adaptability of rootstocks has a fundamental influence on multiple abiotic-stress resistance properties of their scions. Here, we investigated the high-salt resistance of apple rootstocks, by treating 2-year-old apple trees consisting of the ‘Fuji’ cultivar grafted onto three different dwarfing rootstocks (SH6, M9-T337 and G935) with a high-salt solution for 30 days. We found that the high-salt stress caused leaf yellowing and growth inhibition to all apple plants, and that the most serious reduction in relative chlorophyll content, plant height and biomass accumulation was associated with the SH6 rootstock. In addition, based on the salt injury index, the high-salt resistance of the ‘Fuji’ scion on the three dwarfing rootstocks was ranked as G935 > M9-T337 > SH6. In addition, analysis of mineral elemental composition showed that the Na+ content in ‘Fuji’ leaves on the G935 rootstock increased less than twofold compared to the control, whereas those on the SH6 rootstock increased nearly 20-fold, accompanied with reduced K and Fe element absorption under high-salt treatments. Expression analysis of nine Na+ transport-related genes indicated that in the salt overly sensitive (SOS) pathway, the SOS1, SOS2 and SOS3 genes showed considerably greater induction by high-salt stress in G935 roots than in SH6 roots. Given the Na+ content detected in roots of different rootstocks, we propose that more efficient Na+ extracellular efflux in G935 roots, rather than intracellular Na+ compartmentalization, contributed to the improved characteristics of the G935 rootstock under high-salt conditions.

Potassium (K) is one of the most important macronutrients for plant development and growth. The influence mechanism of different potassium stresses on the molecular regulation and metabolites of apple remains largely unknown. In this research, physiological, transcriptome, and metabolite analyses were compared under different K conditions in apple seedlings. The results showed that K deficiency and excess conditions influenced apple phenotypic characteristics, soil plant analytical development (SPAD) values, and photosynthesis. Hydrogen peroxide (H 2 O 2 ) content, peroxidase (POD) activity, catalase (CAT) activity, abscisic acid (ABA) content, and indoleacetic acid (IAA) content were regulated by different K stresses. Transcriptome analysis indicated that there were 2,409 and 778 differentially expressed genes (DEGs) in apple leaves and roots under K deficiency conditions in addition to 1,393 and 1,205 DEGs in apple leaves and roots under potassium excess conditions, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment showed that the DEGs were involved in flavonoid biosynthesis, photosynthesis, and plant hormone signal transduction metabolite biosynthetic processes in response to different K conditions. There were 527 and 166 differential metabolites (DMAs) in leaves and roots under low-K stress as well as 228 and 150 DMAs in apple leaves and roots under high-K stress, respectively. Apple plants regulate carbon metabolism and the flavonoid pathway to respond to low-K and high-K stresses. This study provides a basis for understanding the metabolic processes underlying different K responses and provides a foundation to improve the utilization efficiency of K in apples.

The site of Qujialing experienced a long, sustained process of the development of Neolithic culture in the Jianghan Plain, with a period of some1600 years. Our previous studies based on macrofossil remains and phytoliths revealed that rice ( Oryza sativa ) from Qujialing was already domesticated, and millet ( Setaria italica and Panicum miliaceum ) had also been spread into the site since the Youziling Culture period (5800-5100 BP). Nevertheless, no direct evidence has been provided regarding the daily consumed plant foods, especially plant foods obtained by gathering, throughout the site occupation. This paper thus examines pottery sherds (n=41) associated with culinary practices from Qujialing with starch grain analysis. Apart from starch grains from rice and millet, the results indicate that job’s tears ( Coix lacryma-jobi ), lotus roots ( Nelumbo nucifera ), tubers possibly from Chinese yam ( Dioscorea panthainca ), acorns ( Quercus sp.), and beans ( Vigna sp. or/and Vicia sp.) were consumed by the ancient Qujialing people, within job’s tears and lotus roots were not discovered before in the macrofossil remains and phytoliths. Combining the starch data and multiple lines of evidence from macrofossil remains and phytoliths, it is suggested that rice was among the most frequently consumed plant foods since the first occupation phase at Qujialing, while acorns could have been gradually replaced by other agricultural products (i.e., rice) and became less important food ingredients, especially when agriculture was more developed in the last occupation phase at Qujialing. These novel findings not only complement our previous research by providing the first line of evidence of paleodiet in the Jianghan Plain from the perspective of starch grain analysis but also delivers a better understanding of the characterized dietary trends and agricultural development in the research region.

Mixed farming of rice and millet is one of the basic agricultural modes in the upper and middle Huai River Valley （HRV）. According to the latest data, this agricultural mode appeared during the middle and late Peiligang Culture （7.8-7.0 ka BP） in the upper HRV, and then became a common subsistence economy in the end of the Neolithic （5.04.0 ka BP） in both the upper and middle HRV. However, it is still not clear how this mixed farming developed in the upper HRV after its occurrence, nor are the regional differences in the development of mixed farming between the upper and middle HRV during the Neolithic completely understood. In this paper, flotation and starch analyses were conducted on samples from eight archaeological sites in the upper and middle HRV. The results indicate that the mixed farming of rice and millet first appeared in the later phase of the middle Neolithic in the regions of the Peiligang Culture, then developed quite rapidly in the late Neolithic （6.8-5.0 ka BP）, finally becoming the main subsistence economy at the end of the Neolithic in the upper HRV. However, there are obvious differences in the emergence and development of agriculture between the middle and upper HRV. Rice farming was the only agricultural system during the middle Neolithic, lasting until the end of the Neolithic, when mixed farming appeared in the middle HRV. Furthermore, although mixed farming appeared in both the upper and middle HRV during the end of the Neolithic, the roles of rice, foxtail millet and broomcorn millet in the subsistence economy were not the same. In general, millet was more widely cultivated than rice in the upper HRV, but rice occupied the same or a slightly more prominent position in the middle HRV at the end of the Neolithic. These results are significant for understanding the process of agricultural development and transformation, as well as human adaptation to climatic and cultural variability duringthe Neolithic.

Pogostemon cablin is a medicinally and economically important plant because it contains a high content of patchouli oil. Patchouli oil is composed of more than 24 unique sesquiterpene compounds, with the most abundant being patchouli alcohol (PA). PA is a sesquiterpene and is mainly synthesized through the mevalonate (MVA) pathway in plants in which isopentenyl diphosphate isomerase (IPI) is a key enzyme. Here, a full-length cDNA encoding IPI (designated as PcIPI) was cloned from P. cablin through the rapid amplification of cDNA ends (RACE). Bioinformatics analysis revealed that the full-length PcIPI includes an 879 bp open reading frame (ORF) that encodes a 292 amino acid protein with a theoretical isoelectric point of 5.55 and a molecular mass of 32.8 kDa. Phylogenetic analysis demonstrated that PcIPI shared the most recent genetic relationship with the IPI of Isodon rubescens (86.43%), Salvia miltiorrhiza (85.38%), Plectranthus barbatus (86.54%), and Prunella vulgaris (86.31%). To verify the function of the PcIPI gene in PA biosynthesis, the P. cablin plants were infected with Agrobacterium tumefaciens bearing a pTRV2-PcIPI construct to induce virus-mediated PcIPI silencing. Quantitative real-time polymerase chain reaction (qRT-PCR) and gas chromatography mass spectrometry (GC-MS) analysis were applied to detect changes in the PcIPI catalytic products and transcription levels. The trend of decreasing transcript level was found to be similar to that of decreasing sesquiterpene content. These results revealed that PcIPI silencing had a negative effect on PA biosynthesis.

The archaeological sites of Shunshanji and Hanjing are recorded as the earliest Neolithic sites in the middle Huai river valley, China, which were occupied between 8,500 and 7,500 cal bp, and provide crucial evidence of rice growing. However, the water availability and management of arable land systems for rice cultivation and the main stages of rice processing at the two sites have not been studied before. In this paper, phytolith analyses are discussed in 66 samples from the Shunshanji site and 32 samples from the Hanjing site, with three phases of occupation at each. The ratios of sensitive to fixed forms in the phytolith assemblages over three phases reveal that in Phase I these ratios are relatively low, and they become significantly higher in Phases II and III at both sites. These results suggest that rice was grown probably only using natural water supplies in the lowlands during Phase I, but changed to growing on highly controlled and regularly flooded and drained wetlands in Phase II at the two sites. In addition, seen from the distribution and concentrations of phytoliths from rice, weeds and their by-products, as well as the proportions of each, both double peaked glume cells (DPG) and rice cuneiform bulliform cells (RCB) were found together with some rice paddy weed phytoliths. The concentrations of DPG were much higher than those of RCB in most samples from the Shunshanji and Hanjing sites over the three phases. The findings imply that rice ears together with the stems and leaves were probably stored after the harvest, then threshed and de-husked simultaneously, and the straw and chaff by-products were discarded together at the sites between 8,500 and 7,500 cal BP. The study in this paper aims towards a better understanding of past human behaviour and social organization in rice growing communities during the middle Neolithic in eastern China.

The upper-middle Huai River valley（HRV） is located in the transition zone in the middle of eastern China. Previous studies have shown that the past populations in the upper HRV obtained plant food through gathering and cultivation of both rice and millet during the middle Neolithic. However, for the middle HRV it is not very clear what methods were used by humans to obtain plant food in that time. In this paper, starch grain analysis was carried out on 17 stone tools and 29 cauldron pottery sherds unearthed at Shunshanji（8.5–7.0 kyr BP）, which is known as the earliest Neolithic site in the middle HRV excavated so far. Here, ancient starches from Coix lacryma-jobi, Triticeae, Oryza sativa, Trichosanthes kirilowii and one unidentified specie were recovered. This study contributes to the limited knowledge of food strategies as observed in the middle HRV. It demonstrates that both gathering and cultivation of rice took place during the middle-Neolithic. Among the identified species, Coix lacryma-jobi appears to have been the main plant food at Shunshanji. Moreover, it is clear that Oryza sativa was not consumed as much as other plant species according to the occurrence frequency of different starch grains. Starch grains were also found on the used surfaces of grinding stone implements as well as pestles, which means that these stone tools were used for food processing. In addition, axes from Shunshanji may also have been used for food processing as well as wood working because starches were also found on the edge of axes. Information about subsistence strategies and tool use at Shunshanji will also be helpful to understanding the utilization of plants and agricultural development in the middle HRV during the early-middle Neolithic period.

In the central plain of China, grinding tools are a common category of artefacts at sites attributed to the Peiligang Culture (c. 9000-7000 BP). This paper focuses on the grinding tool assemblage from the site of Tanghu, the largest Peiligang Culture settlement yet discovered. The results from the microwear and residue analyses both suggest that cereals were the primary plant material processed with the grinding tools. Other plants, including acorns and underground storage organs, were also processed, but probably to a smaller extent. Furthermore, microwear analysis suggests that the dry-grinding technique was adopted for cereal processing, and a piece of hide or animal skin was placed underneath the grinding slabs to gather the processed plant material. Apart from plant food processing, one of the grinding tools was also involved in processing bone. These data put more insights into the Neolithic culinary practices and different uses of grinding tools in this region.