Integrative Morphological, Physiological, Proteomics Analyses of Jujube Fruit Development Provide Insights Into Fruit Quality Domestication From Wild Jujube to Cultivated Jujube

Jujube (Ziziphus jujuba) was domesticated from wild jujube (Z. jujuba var. spinosa). Here, integrative physiological, metabolomic, and comparative proteomic analyses were performed to investigate the fruit expansion and fruit taste components in a jujube cultivar ‘Junzao’ and a wild jujube ‘Qingjiansuanzao’ with contrasting fruit size and taste. We revealed that the duration of cell division and expansion largely determined the final fruit size, while the intercellular space in the mesocarp dictated the ratio of mesocarp volume in mature fruits. The high levels of endogenous gibbereline3 (GA) and zeatin in the growing fruit of ‘Junzao’ were associated with their increased fruit expansion. Compared with ‘Junzao,’ wild jujube accumulated lower sugars and higher organic acids. Furthermore, several protein co-expression modules and important member proteins correlated with fruit expansion, sugar synthesis, and ascorbic acid metabolism were identified. Among them, GA20OX involved in GA biosynthesis was identified as a key protein regulating fruit expansion, whereas sucrose-6-phosphate synthase (SPS) and neutral invertase (NINV) were considered as key enzymes promoting sugar accumulation and as major factors regulating the ratio of sucrose to hexose in jujube fruits, respectively. Moreover, the increase of Nicotinamide adenine dinucleotide-Malate dehydrogenase (NAD-MDH) activity and protein abundance were associated with the malic acid accumulation, and the high accumulation of ascorbic acid in wild jujube was correlated with the elevated abundance of GalDH, ZjAPXs, and MDHAR1, which are involved in the ascorbic acid biosynthesis and recycling pathways. Overall, these results deepened the understanding of mechanisms regulating fruit expansion and sugar/acids metabolisms in jujube fruit.

Effects of Elevated CO2 on Photosynthetic Accumulation, Sucrose Metabolism-Related Enzymes, and Genes Identification in Goji Berry (Lycium barbarum L.)

Goji berry (Lycium barbarum L.) exposure to elevated CO2 (eCO2) for long periods reduces their sugar and secondary metabolite contents. However, sugar accumulation in fruit depends on photosynthesis and photoassimilate partitioning. This study aimed to explore photosynthesis, sugar content, and sucrose metabolism-related enzyme activities in goji berry leaves and fruits under ambient and eCO2 levels, and identify the genes encoding L. barbarum acid invertase (LBAI), L. barbarum sucrose synthase (LBSS), L. barbarum sucrose phosphate synthase (LBSPS), and L. barbarum neutral invertase (LBNI), based on transcriptome profiling. Further, the characterization of four identified genes was analyzed including subcellular localization and expression patterns. In plants grown under eCO2 for 90 or 120 days, the expression of the above-mentioned genes changed significantly as the photosynthetic rate increased. In addition, leaf and fruit sugar contents decreased, and the activities of four sucrose metabolism-related enzymes increased in leaves, while acid and neutral invertase increased in fruits. Protein sequence analysis demonstrated that LBAI and LBNI contain a conservative structure domain belonging to the glycosyl hydrolases (Glyco_hydro) family, and both LBSS and LBSPS belonging to the sucrose synthase (Sucrose_synth) and glycosyltransferase (Glycos_transf) family. Subcellular localization analysis showed that LBAI, LBNI, and LBSS were all located in the nucleus, plasma membrane, and cytoplasm, while LBSPS was located in the plasma membrane. The expressions of LBAI, LBSPS, and LBNI were high in the stems, whereas LBSS was predominantly expressed in the fruits. Our findings provide fundamental data on photosynthesis and sugar accumulation trends in goji berries under eCO2 exposure.

Effect of Elevated CO2 on Photosynthetic Accumulation and Sucrose Metabolism-related Enzyme Activity and Gene Expression Patterns in Goji Berry (Lycium Barbarum L.)

Background: Exposing goji berries to elevated CO2 (eCO2) for long periods reduces sugar and secondary metabolite contents. However, sugar accumulation in fruit depends on photosynthesis and photoassimilate partitioning. The objectives of this study were a) to explore photosynthesis, sugar content, and sucrose metabolism-related enzyme activity in goji berry leaves and fruits under ambient and elevated CO2, and b) to identify the genes encoding Lycium barbarum acid invertase (LBAI), L. barbarum sucrose synthase (LBSS), L. barbarum sucrose phosphate synthase (LBSPS), and L. barbarum neutral invertase (LBNI) based on transcriptome profiling and expression analyses in different tissues. Results: The results showed that the expression of the above genes changed significantly in plants grown under eCO2 for 90 and 120 days as photosynthetic rate increased, while leaf and fruit sugar content decreased and the activity of four sucrose metabolism-related enzymes increased in leaves, and acid and neutral invertase increased in fruits. Furthermore, a significant correlation was detected between sugar content and enzyme activity. The expression of LBAI, LBSPS, and LBNI were high in stems, whereas LBSS was predominantly expressed in fruits. Conclusions: eCO2 affects chlorophyll, photosynthesis, sugars, and related enzyme activities in goji berry leaves and fruits after 90 and 120 days of treatment. Our findings provide fundamental data on photosynthesis and sugar accumulation trends in goji berries under eCO2 exposure.

Activity invertase and amylase in Marandu grass under shading and nitrogen fertilization

The objective of this study was to evaluate the activity of invertases and amylases in Brachiaria brizantha cv. Marandu under various shade and nitrogen fertilization conditions. The experiment was carried out in a greenhouse using a 4 x 2 factorial scheme (shading levels of 0, 30, 50, and 80% and fertilization with 0 and 100 kg N ha-1). The activity of the enzymes, cytosol-neutral invertase (Inv-N), vacuole acid (Inv-V) and cell-acidic acid (Inv-CW), reducing sugars (RS), and α and β-amylases were evaluated (α = 0.05). The interaction was significant for Inv-N within the leaf. In the first cycle, the highest activity was in fertilized plants with 30, 50, and 80% shading. For Inv-CW in the 1st cycle, the highest activity occurred with 0, 30, and 50% shading. However, the interaction for Inv-V leaf activity was not significant in the 1st and 2nd cycles. The highest activity observed for Inv-V was in the fertilized plants, suggesting that fertilization increased the enzymatic activity. The activity of the invertases increased both under 30-50% shaded conditions and in full sun. Furthermore, invertase activity was directly linked to the osmoregulatory system. The reduction in RS was related to a low photosynthetic rate, and an increase α and β-amylase was associated with the use of reserve energy sources to meet energetic needs.

Crystal structure of Arabidopsis thaliana neutral invertase 2

The metabolism of sucrose is of crucial importance for life on Earth. In plants, enzymes called invertases split sucrose into glucose and fructose, contributing to the regulation of metabolic fluxes. Invertases differ in their localization and pH optimum. Acidic invertases present in plant cell walls and vacuoles belong to glycoside hydrolase family 32 (GH32) and have an all-β structure. In contrast, neutral invertases are located in the cytosol and organelles such as chloroplasts and mitochondria. These poorly understood enzymes are classified into a separate GH100 family. Recent crystal structures of the closely related neutral invertases InvA and InvB from the cyanobacterium Anabaena revealed a predominantly α-helical fold with unique features compared with other sucrose-metabolizing enzymes. Here, a neutral invertase (AtNIN2) from the model plant Arabidopsis thaliana was heterologously expressed, purified and crystallized. As a result, the first neutral invertase structure from a higher plant has been obtained at 3.4 Å resolution. The hexameric AtNIN2 structure is highly similar to that of InvA, pointing to high evolutionary conservation of neutral invertases.

Altered sucrose metabolism and gene regulation in wheat (Triticum aestivum) K-type cytoplasmic male sterility

K-Type cytoplasmic male sterility (K-CMS) plays an important role in breeding hybrid wheat. This study was designed to investigate the association of sucrose metabolism with K-CMS in wheat (Triticum aestivum L.) anthers at the binucleate stage. Levels of sucrose in the anthers of the K-CMS line remained higher than in the fertile line, but glucose and fructose contents in the anthers of the K-CMS line were dramatically lower than in the fertile line. Compared with the fertile line, the activities of cell-wall-bound invertase (CWIN), neutral invertase and vacuolar invertase (VIN) were significantly reduced. Quantitative real-time polymerase chain reaction analyses showed that the expression levels of one CWIN gene (IVR1), one VIN gene (IVR5) and a sucrose transporter gene (TaSUT1) were significantly downregulated in K-CMS anthers. Furthermore, western blot confirmed that the protein expression level of IVR1 was higher in sterile anthers than in male fertile anthers. Thus, it appears that the accumulation of sucrose in K-CMS anthers might involve a decrease in activity and a reduction in content of invertase. In conclusion, the results suggest that an inability to metabolise incoming sucrose to hexoses may be involved in the K-CMS pollen-developmental lesion.