Collins Ogutu

Comparative assessment of sugar and malic acid composition in cultivated and wild apples

Soluble sugar and malic acid contents in mature fruits of 364 apple accessions were quantified using high-performance liquid chromatography (HPLC). Fructose and sucrose represented the major components of soluble sugars in cultivated fruits, whilst fructose and glucose were the major items of sugars in wild fruits. Wild fruits were significantly more acidic than cultivated fruits, whilst the average concentration of total sugars and sweetness index were quite similar between cultivated and wild fruits. Thus, our study suggests that fruit acidity rather than sweetness is likely to have undergone selection during apple domestication. Additionally, malic acid content was positively correlated with glucose content and negatively correlated with sucrose content. This suggests that selection of fruit acidity must have an effect on the proportion of sugar components in apple fruits. Our study provides information that could be helpful for future apple breeding.

Genes Encoding Aluminum-Activated Malate Transporter II and their Association with Fruit Acidity in Apple

A gene encoding aluminum‐activated malate transporter (ALMT) was previously reported as a candidate for the Ma locus controlling acidity in apple (Malus × domestica Borkh.). In this study, we found that apple ALMT genes can be divided into three families and the Ma1 gene belongs to the ALMTII family. Duplication of ALMTII genes in apple is related to the polyploid origin of the apple genome. Divergence in expression has occurred between the Ma1 gene and its homologs in the ALMTII family and only the Ma1 gene is significantly associated with malic acid content. The Ma locus consists of two alleles, Ma1 and ma1. Ma1 resides in the tonoplast and its ectopic expression in yeast was found to increase the influx of malic acid into yeast cells significantly, suggesting it may function as a vacuolar malate channel. In contrast, ma1 encodes a truncated protein because of a single nucleotide substitution of G with A in the last exon. As this truncated protein resides within the cell membrane, it is deemed to be nonfunctional as a vacuolar malate channel. The frequency of the Ma1Ma1 genotype is very low in apple cultivars but is high in wild relatives, which suggests that apple domestication may be accompanied by selection for the Ma1 gene. In addition, variations in the malic acid content of mature fruits were also observed between accessions with the same genotype in the Ma locus. This suggests that the Ma gene is not the only genetic determinant of fruit acidity in apple.

Copy number variation of a gene cluster encoding endopolygalacturonase mediates flesh texture and stone adhesion in peach

Highlight Copy number variation at the F-M locus plays a driving role in flesh texture diversification in peach.

Evolutionary origin of the NCSI gene subfamily encoding norcoclaurine synthase is associated with the biosynthesis of benzylisoquinoline alkaloids in plants.

Sacred lotus is rich in biologically active compounds, particularly benzylisoquinoline alkaloids (BIAs). Here, we report on isolation of genes encoding (S)-norcoclaurine synthase (NCS) in sacred lotus, which is a key entry-enzyme in BIA biosynthesis. Seven NCS genes, designated NnNCS1 through NnNCS7, were identified in the sacred lotus genome, and five are located next to each other within a 83 kb region on scaffold 8. The NCS genes are divided into two subfamilies, designated NCSI and NCSII. The NCSII genes are universal in plants, while the NCSI genes are only identified in a limited number of dicotyledonous taxa that produce BIAs. In sacred lotus, only NnNCS4 belongs to the NCSII subfamily, whilst the rest NCS genes within the NCSI subfamily. Overall, the NnNCS7 gene was predominantly expressed in all tested tissues, and its expression is significantly correlated with alkaloid content in leaf. In contrast, the NnNCS4 expression shows no significant correlation with alkaloid accumulation in leaf, and its lack of expression cannot inhibit alkaloid accumulation. Taken together, these results suggest that the NCSI subfamily is crucial for BIA biosynthesis, and its origin may represent an important evolutionary event that allows certain plant taxa to produce BIAs.

Analysis of Isoquinoline Alkaloid Composition and Wound-Induced Variation in Nelumbo Using HPLC-MS/MS.

Alkaloids are the most relevant bioactive components in lotus, a traditional herb in Asia, but little is known about their qualitative and quantitative distributions. Here, we report on the alkaloid composition in various lotus organs. Lotus laminae and embryos are rich in isoquinoline alkaloids, whereas petioles and rhizomes contain trace amounts of alkaloids. Wide variation of alkaloid accumulation in lamina and embryo was observed among screened genotypes. In laminae, alkaloid accumulation increases during early developmental stages, reaches the highest level at full size stage, and then decreases slightly during senescence. Vegetative and embryogenic tissues accumulate mainly aporphine-type and bisbenzylisoquinoline-type alkaloids, respectively. Bisbenzylisoquinoline-type alkaloids may be synthesized mainly in lamina and then transported into embryo via latex through phloem translocation. In addition, mechanical wounding was shown to induce significant accumulation of specific alkaloids in lotus leaves.

Two amino acid changes in the R3 repeat cause functional divergence of two clustered MYB10 genes in peach

R2R3-MYB genes play a pivotal role in regulating anthocyanin accumulation. Here, we report two tandemly duplicated R2R3-MYB genes in peach, PpMYB10.1 and PpMYB10.2, with the latter showing lower ability to induce anthocyanin accumulation than the former. Site-directed mutation assay revealed two amino acid changes in the R3 repeat, Arg/Lys66 and Gly/Arg93, responsible for functional divergence between these two PpMYB10 genes. Anthocyanin-promoting activity of PpMYB10.2 was significantly increased by a single amino acid replacement of Arg93 with Gly93. However, either the Gly93 → Arg93 or Arg66 → Lys66 substitutions alone showed little impact on anthocyanin-promoting activity of PpMYB10.1, but simultaneous substitutions caused a significant decrease. Reciprocal substitution of Arg/Gly93 could significantly alter binding affinity to PpbHLH3, while the Arg66 → Lys66 substitution is predicted to affect the folding of the MYB DNA-binding domain, instead of PpbHLH3-binding affinity. Overall, the change of anthocyanin-promoting activity was accompanied with that of bHLH-binding affinity, suggesting that DNA-binding affinity of R2R3-MYBs depends on their bHLH partners. Our study is helpful for understanding of functional evolution of R2R3-MYBs and their interaction with DNA targets.

A Ma10 gene encoding P‐type ATPase is involved in fruit organic acid accumulation in apple

Summary Acidity is one of the main determinants of fruit organoleptic quality. Here, comparative transcriptome analysis was conducted between two cultivars that showed a significant difference in fruit acidity, but contained homozygous non‐functional alleles at the major gene Ma1 locus controlling apple fruit acidity. A candidate gene for fruit acidity, designated M10, was identified. The M10 gene encodes a P‐type proton pump, P3A‐ATPase, which facilitates malate uptake into the vacuole. The Ma10 gene is significantly associated with fruit malate content, accounting for ~7.5% of the observed phenotypic variation in apple germplasm. Subcellular localization assay showed that the Ma10 is targeted to the tonoplast. Overexpression of the Ma10 gene can complement the defect in proton transport of the mutant YAK2 yeast strain and enhance the accumulation of malic acid in apple callus. Moreover, its ectopic expression in tomato induces a decrease in fruit pH. These results suggest that the Ma10 gene has the capacity for proton pumping and plays an important role in fruit vacuolar acidification in apple. Our study provides useful knowledge towards comprehensive understanding of the complex mechanism regulating apple fruit acidity.

Activator‐type R2R3‐MYB genes induce a repressor‐type R2R3‐MYB gene to balance anthocyanin and proanthocyanidin accumulation

Anthocyanin and proanthocyanidin (PA) accumulation is regulated by both myeloblastosis (MYB) activators and repressors, but little information is available on hierarchical interactions between the positive and negative regulators. Here, we report on a R2R3-MYB repressor in peach, designated PpMYB18, which acts as a negative regulator of anthocyanin and PA accumulation. PpMYB18 can be activated by both anthocyanin- and PA-related MYB activators, and is expressed both at fruit ripening and juvenile stages when anthocyanins or PAs, respectively, are being synthesized. The PpMYB18 protein competes with MYB activators for binding to basic Helix Loop Helixes (bHLHs), which develops a fine-tuning regulatory loop to balance PA and anthocyanin accumulation. In addition, the bHLH binding motif in the R3 domain and the C1 and C2 repression motifs in the C-terminus of PpMYB18 both confer repressive activity of PpMYB18. Our study also demonstrates a modifying negative feedback loop, which prevents cells from excess accumulation of anthocyanin and PAs, and serves as a model for balancing secondary metabolite accumulation at the transcriptional level.

Investigation of benzylisoquinoline alkaloid biosynthetic pathway and its transcriptional regulation in lotus

Lotus predominantly accumulates benzylisoquinoline alkaloids (BIAs), but their biosynthesis and regulation remain unclear. Here, we investigated structural and regulatory genes involved in BIA accumulation in lotus. Two clustered CYP80 genes were identified to be responsible for the biosynthesis of bis-BIAs and aporphine-type BIAs, respectively, and their tissue-specific expression causes divergence in alkaloid component between leaf and embryo. In contrast with the common (S)-reticuline precursor for most BIAs, aporphine alkaloids in lotus leaf may result from the (S)-N-methylcoclaurine precursor. Structural diversity of BIA alkaloids in the leaf is attributed to enzymatic modifications, including intramolecular C–C phenol coupling on ring A and methylation and demethylation at certain positions. Additionally, most BIA biosynthetic pathway genes show higher levels of expression in the leaf of high-BIA cultivar compared with low-BIA cultivar, suggesting transcriptional regulation of BIA accumulation in lotus. Five transcription factors, including three MYBs, one ethylene-responsive factor, and one basic helix–loop–helix (bHLH), were identified to be candidate regulators of BIA biosynthesis in lotus. Our study reveals a BIA biosynthetic pathway and its transcriptional regulation in lotus, which will enable a deeper understanding of BIA biosynthesis in plants.Synthesis of medicinal compounds in lotusResearchers at the Chinese Academy of Sciences have discovered the genes in lotus responsible for the synthesis of benzylisoquinole alkaloids (BIAs), a diverse set of pharmacologically significant plant metabolites. By comparing gene expression in two lotus varieties, a high- and low-BIA cultivar, they identified the genes that were active during BIA biosynthesis as well as five transcription factors which regulate the process. One of the biosynthesis genes is expressed more strongly in the embryo and another in the leaves, leading to the accumulation of different types of BIAs in these tissues. Although many of these genes are related to known BIA synthesis genes in other species, identifying the lotus equivalents not only reveals the particulars of the process in this species but also expands our understanding of BIA synthesis in general.