Melatonin is a potent naturally occurring reactive oxygen species (ROS) and reactive nitrogen species (RNS) scavenger in plants. Melatonin protects plants from oxidative stress and, therefore, it improves their tolerance against a variety of environmental abiotic stressors. N-acetylserotonin-O-methyltransferase (ASMT) is a specific enzyme required for melatonin synthesis. In this report, an ASMT gene was cloned from apple rootstock (Malus zumi Mats) and designated as MzASMT1 (KJ123721). The MzASMT1 expression was induced by drought stress in apple leaves. The upregulation of MzASMT1 in the apple leaf positively relates to melatonin production over a 24-hr dark/light cycle. Purified MzASMT1 protein expressed in E. coli converted its substrates to melatonin with an activity of approximately 5.5 pmol/min/mg protein. The transient transformation in tobacco identified that MzASMT1 is located in cytoplasm of the cell. When MzASMT1 gene driven by 35S promoter was transferred to Arabidopsis, melatonin levels in transgenic Arabidopsis plants were 2-4 times higher than those in the wild type. The transgenic Arabidopsis plants had significantly lower intrinsic ROS than the wild type and therefore these plants exhibited greater tolerance to drought stress than that of wild type. This is, at least partially, attributed to the elevated melatonin levels resulting from the overexpression of MzASMT1. The results elucidated the important role that membrane-located melatonin synthase plays in drought tolerance. These findings have significant implications in agriculture.

Within the chloroplasts reactive oxygen species (ROS) are generated during photosynthesis and stressful conditions. Excessive ROS damages chloroplasts and reduces photosynthesis if not properly detoxified. In this current study, we document that chloroplasts produce melatonin, a recentlydiscovered plant antioxidant molecule. When N-acetylserotonin, a substrate for melatonin synthesis, was fed to purified chloroplasts, they produced melatonin in a dose-response manner. To further confirm this function of chloroplasts, the terminal enzyme for melatonin synthesis, N-acetylserotonin-O-methyltransferase (ASMT), was cloned from apple rootstock, Malus zumi. The in vivo fluorescence observations and Western blots confirmed MzASMT9 was localized in the chloroplasts. A study of enzyme kinetics revealed that the K m and V max of the purified recombinant MzASMT9 protein for melatonin synthesis were 500 μM and 12 pmol/min·mg protein, respectively. Arabidopsis ectopicallyexpressing MzASMT9 possessed improved melatonin level. Importantly, the MzASMT9 gene was found to be upregulated by high light intensity and salt stress. Increased melatonin due to the highlyexpressed MzASMT9 resulted in Arabidopsis lines with enhanced salt tolerance than wild type plants, as indicated by reduced ROS, lowered lipid peroxidation and enhanced photosynthesis. These findings have agricultural applications for the genetic enhancement of melatonin-enriched plants for increasing crop production under a variety of unfavorable environmental conditions.Chloroplasts are critically important plant cellular organelles with the major function of performing photosynthesis. The chlorophyll pigments in chloroplasts capture energy from photons, with this energy being used to synthesize organic molecules from carbon dioxide via the Calvin cycle. Large quantities of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated during photosynthesis, especially under the stressful conditions such as exposure to excessive light intensity, heat, salt, cold, drought and environmental pollutants 1-4 . ROS and RNS, if not properly detoxified, damages chloroplasts and reduces their photosynthetic efficiency, ultimately killing the cell. This damage is referred to as oxidative stress. During evolution, plants developed an array of mechanisms to protect themselves against oxidative stress 5 . One mechanism is the production of antioxidants; which include ascorbic acid, carotenoids, tocopherol, glutathione and polyphenols 6 . Interestingly another antioxidant, melatonin, initially thought to be exclusively an animal hormone, was identified in plants in 1995 7,8 . Since then melatonin has been detected in many different species of plants and plant products [9][10][11][12][13] . The significance of melatonin in plants is related to its potent free radical scavenging and antioxidant capacity [14][15][16][17] . In contrast to several other antioxidants, melatonin can enter every sub-cellular compartment due to its amphiphilic nature 16 . In addition, melaton...

Advances in high-throughput SNP genotyping and genome sequencing technologies have enabled genome-wide association mapping in dissecting the genetic basis of complex quantitative traits. In this study, 82 SSRs and 884 SNPs with minor allele frequencies (MAF) over 0.20 were used to compare their ability to assess population structure, principal component analysis (PCA) and relative kinship in a maize association panel consisting of 154 inbred lines. Compared to SNPs, SSRs provided more information on genetic diversity. The expected heterozygosity (He) of SSRs and SNPs averaged 0.65 and 0.44, and the polymorphic information content of these two markers was 0.61 and 0.34 in this panel, respectively. Additionally, SSRs performed better at clustering all lines into groups using STRUCTURE and PCA approaches, and estimating relative kinship. For both marker systems, the same clusters were observed based on PCA and the first two eigenvectors accounted for similar percentage of genetic variations in this panel. The correlation coefficients of each eigenvector from SSRs and SNPs decreased sharply when the eigenvector varied from 1 to 3, but kept around 0 when the eigenvector were over 3. The kinship estimates based on SSRs and SNPs were moderately correlated (r (2) = 0.69). All these results suggest that SSR markers with moderate density are more informative than SNPs for assessing genetic relatedness in maize association mapping panels.

Adventitious root formation is essential for the vegetative propagation of perennial woody plants. During the juvenile-to-adult phase change mediated by the microRNA156 (miR156), the adventitious rooting ability decreases dramatically in many species, including apple rootstocks. However, the mechanism underlying how miR156 affects adventitious root formation is unclear. In the present study, we showed that in the presence of the synthetic auxin indole-3-butyric acid (IBA), semi-lignified leafy cuttings from juvenile phase (Mx-J) and rejuvenated (Mx-R) Malus xiaojinensis trees exhibited significantly higher expression of miR156, PIN-FORMED1 (PIN1), PIN10, and rootless concerning crown and seminal roots-like (RTCS-like) genes, thus resulting in higher adventitious rooting ability than those from adult phase (Mx-A) trees. However, the expression of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE26 (SPL26) and some auxin response factor (ARF) gene family members were substantially higher in Mx-A than in Mx-R cuttings. The expression of NbRTCS-like but not NbPINs and NbARFs varied with miR156 expression in tobacco (Nicotiana benthamiana) plants transformed with 35S:MdMIR156a6 or 35S:MIM156 constructs. Overexpressing the miR156-resistant MxrSPL genes in tobacco confirmed the involvement of MxSPL20, MxSPL21&22, and MxSPL26 in adventitious root formation. Together, high expression of miR156 was necessary for auxin-induced adventitious root formation via MxSPL26, but independent of MxPINs and MxARFs expression in M. xiaojinensis leafy cuttings.

Many efforts have been made to map quantitative trait loci (QTLs) to facilitate practical marker-assisted selection (MAS) in plants. In the present study, using MapQTL and BSA-seq (bulk segregant analysis using next generation sequencing) with two independent pedigree-based populations, we identified four major genome-wide QTLs responsible for apple fruit acidity. Candidate genes were screened in major QTL regions, and three functional gene markers, including a non-synonymous A/G single-nucleotide polymorphism (SNP) in the coding region of MdPP2CH, a 36-bp insertion in the promoter of MdSAUR37 and a previously reported SNP in MdALMTII, were validated to influence the malate content of apple fruits. In addition, MdPP2CH inactivated three vacuolar H -ATPases (MdVHA-A3, MdVHA-B2 and MdVHA-D2) and one aluminium-activated malate transporter (MdALMTII) via dephosphorylation and negatively influenced fruit malate accumulation. The dephosphotase activity of MdPP2CH was suppressed by MdSAUR37, which implied a higher hierarchy of genetic interaction. Therefore, the MdSAUR37/MdPP2CH/MdALMTII chain cascaded hierarchical epistatic genetic effects to precisely determine apple fruit malate content. An A/G SNP (-1010) on the MdMYB44 promoter region from a major QTL (qtl08.1) was closely associated with fruit malate content. The predicted phenotype values (PPVs) were estimated using the tentative genotype values of the gene markers, and the PPVs were significantly correlated with the observed phenotype values. Our findings provide an insight into plant genome-based selection in apples and will aid in conducting research to understand the fundamental physiological basis of quantitative genetics.