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

Abstract

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.

Keywords: Ziziphus jujuba; ascorbic acid; fruit expansion; gibberellin; proteomics.

FIGURE 1

The fruit growth pattern of cultivated jujube ‘Junzao’ and wild jujube ‘Qingjiansuanzao’. (A) Fruit ripening progress of ‘Junzao’ and ‘Qingjiansuanzao’ represented by five stages at 20, 40, 60,80, and 100 days after flowering (DAF); (B) Fruit fresh weight increase patterns; (C) Cross-section of fruits sampled weekly from 7 DAF to 49 DAF; (D) Anatomy observation of the mesocarp from 7 to 49 DAF and 77 DAF; (E–G) indicate the change in the pattern of the cell layer, cell area, and intercellular space of the mesocarp from 7 to 49 DAF weekly and at 77 DAF. Values are means of three biological replicates ± SD.

FIGURE 2

Dynamics of phytohormones IAA, ZT, GA₃, and ABA during fruit development and ripening in jujube cultivar ‘Junzao’ and wild jujube ‘Qingjiansuanzao’. Values are means of three biological replicates ± SD. * and ** indicate significant differences at levels of p < 0.05 and p < 0.01 by t-test, respectively.

FIGURE 3

Dynamics of sugar, organic acids, and related enzyme activities during fruit development and ripening. (A) Changes in contents of sucrose, fructose, glucose, malic acid, citric acid, quincy acid, and succinic acid during fruit development in ‘Junzao’ and ‘Qingjiansuanzao’; (B) Activity changes of key enzymes involved in sugar and organic acid metabolism. Values are means of three biological replicates ± SD. * and ** indicate significant differences between the two accessions at levels p < 0.05 and p < 0.01 by t-test, respectively.

FIGURE 4

Proteomic profile and protein co-expression network analysis during the fruit development of ‘Junzao’ and ‘Qingjiansuanzao’. (A) Heatmap representation of protein expression profile during fruit development. J20–J100 and S20–S100 represent 20, 40, 60, 80, and 100 DAF of ‘Junzao’ and ‘Qingjiansuanzao,’ respectively. (B) Number of differential expressed proteins (DEPs) detected between neighbor stages and between ‘Junzao’ (indicated by a blue inner circle) and ‘Qingjiansuanzao’ (orange outer circle) at the same stage. (C) Hierarchical cluster tree of co-expression modules identified by weighted gene co-expression network analysis (WGCNA). Each leaf in the tree represents one protein and each tree branch stands for a module. (D) Module–fruit developmental stage associations. The color of each cell at the row–column intersection indicates the correlation coefficient r (up) between the modules (row) and the protein expression profile at a fruit developmental stage (column) and the r-associated significance p-value (down). The WGCNA network of top hub genes in modules Yellow (E), Lightyellow (F), and Black (G). PGDH, D-3-phosphoglycerate dehydrogenase; ACG, glucose-6-phosphate 1-dehydrogenase; SBP, selenium-binding protein 2; SPS, sucrose-phosphate synthase; KAB, voltage-gated potassium channel; PAT, bifunctional aspartate aminotransferase and glutamate; GPX6, phospholipid hydroperoxide glutathione peroxidase 6; LACS2, long chain acyl-CoA synthetase 2; PGIC1, glucose-6-phosphate isomerase; ALDH3H1, aldehyde dehydrogenase family 3 member H1; CCR1, cinnamoyl-CoA reductase 1; APX, ascorbate peroxidase.

FIGURE 5

Accumulation profile of proteins associated with gibberellin (GA) and cell division and expansion. (A) Expression of identified proteins involved in GA biosynthesis and signal transduction. (B) Expression pattern of proteins related to cell production and cell wall modification.

FIGURE 6

Expression profile of proteins involved in sugar and organic acid metabolism. (A) Pathway of sugar metabolism and transport in jujube fruit. (B) Expression profile of proteins related to sugar metabolism and transport in the fruits of ‘Junzao’ and ‘Qingjiansuanzao’ from 20 DAF to 100 DAF. (C) Expression profile of proteins involved in organic acid metabolism related to glycolysis and TCA cycle in the fruits of ‘Junzao’ and ‘Qingjiansuanzao’ from 20 DAF to 100 DAF . Note that only a subset of primary proteins required for soluble sugar and starch metabolism and transportation in jujube fruits is displayed. The heatmaps were drawn using log₂-transformed protein abundance. Abbreviations of enzymes for sugar metabolism and transportation: FK, fructokinase; F6P, fructose-6-phosphate; Fru, D-fructose; Glc, D-glucose; G6P, glucose-6-phosphate; NINV, neutral invertase; SPP, sucrose-phosphate phosphatase; SPS, sucrose-phosphate synthase; SUC, sucrose carrier or transporter; SUSY, sucrose synthase; UDPG, UDP-D-glucose; AINV, vacuolar acid invertase; VGT, vacuole glucose transporter; ERD6, ERD6-like transporters. STP, Sugar Transport Protein; SUC, Sucrose transporter; TMT, tonoplast membrane transporter. Abbreviations of enzymes for TCA cycle and cytoplasmic malate metabolism: PK, pyruvate kinase; PDC, pyruvate dehydrogenase complex; PDH, pyruvate dehydrogenase; CS, citrate synthase; IDH, isocitrate dehydrogenase; OGDH, 2-oxoglutarate dehydrogenase; SCS, succinyl-CoA synthetase; SDH, succinate dehydrogenase; FUM, Fumarase; cMDH, cytosolic malate dehydrogenase; NAD-ME, NAD-malic enzyme; NADP-ME, NADP-malic enzyme; PEPC, phosphoenolpyruvate carboxylase.

FIGURE 7

Expression profile of proteins involved in ascorbic acid (AsA) metabolism and the dynamics of AsA contents during fruit development and ripening. (A) Protein expression changes involved in AsA biosynthesis and recycling pathways in jujube fruits. (B) Cluster analysis of proteins involved in AsA metabolism. (C) The AsA contents in jujube fruit development. GMP, GDP-d-mannosepyrophosphorylase; GME, GDP-mannose-3′-5′-epimerase; GGP, GDP-l-galactosetransferase; GPP, l-galactose-1-phosphatephosphatase; GalDH, L-galactose dehydrogenase; GalLDH, L-galactono-1,4-lactone dehydrogenase; APX, ascorbate peroxidase; AO, ascorbate oxidase; DHAR, dehydroascorbate reductase; MDHAR, monodehydroascorbate reductase.