Transcriptome Analysis Reveals Genes Respond to Chlorophyll Deficiency in Green and Yellow Leaves of Chrysanthemum morifolium Ramat

Chlorophyll is vital for photosynthesis to produce sugars and other useful biochemical products in green plants. However, the molecular effects of chlorophyll deficiency in Chrysanthemum are largely unknown. In this study, we identified a bud sport mutant chrysanthemum belonging to the variety ‘Nannong Binyun’, which has yellow branches. Plant physiological studies have shown that the yellow color is revealed due to chlorophyll loss. RNA extracts of yellow and green tissues were analyzed using high-throughput RNA-sequencing, and a total of 11,649 tissue enriched unigenes that respond to chlorophyll deficiency were identified, including 4803 unigenes upregulated in yellow tissues and 6846 unigenes in green tissues. GO analysis revealed that these tissue-enriched genes may involve in the physiological processes of chlorophyll accumulation and photosynthesis. In addition, many DEGs from the families of AP2-EREBP, bHLH, MYB, and FAR1 that are associated with plant development and stress response were detected. Our study found that most of the genes from the GRAS family were downregulated in yellow leaves, indicating their putative roles in stem cell maintenance and possible contribution to leaf size determination.

Selection and Evaluation of a Thornless and HLB-Tolerant Bud-Sport of Pummelo Citrus With an Emphasis on Molecular Mechanisms

The selection of elite bud-sports is an important breeding approach in horticulture. We discovered and evaluated a thornless pummelo bud-sport (TL) that grew more vigorously and was more tolerant to Huanglongbing (HLB) than the thorny wild type (W). To reveal the underlying molecular mechanisms, we carried out whole-genome sequencing of W, and transcriptome comparisons of W, TL, and partially recovered thorny “mutants” (T). The results showed W, TL, and T varied in gene expression, allelic expression, and alternative splicing. Most genes/pathways with significantly altered expression in TL compared to W remained similarly altered in T. Pathway and gene ontology enrichment analysis revealed that the expression of multiple pathways, including photosynthesis and cell wall biosynthesis, was altered among the three genotypes. Remarkably, two polar auxin transporter genes, PIN7 and LAX3, were expressed at a significantly lower level in TL than in both W and T, implying alternation of polar auxin transport in TL may be responsible for the vigorous growth and thornless phenotype. Furthermore, 131 and 68 plant defense-related genes were significantly upregulated and downregulated, respectively, in TL and T compared with W. These genes may be involved in enhanced salicylic acid (SA) dependent defense and repression of defense inducing callose deposition and programmed cell death. Overall, these results indicated that the phenotype changes of the TL bud-sport were associated with tremendous transcriptome alterations, providing new clues and targets for breeding and gene editing for citrus improvement.

Transcriptomic and Weighted Gene Co-expression Correlation Network Analysis Reveal Resveratrol Biosynthesis Mechanisms Caused by Bud Sport in Grape Berry

Resveratrol is a natural polyphenol compound produced in response to biotic and abiotic stresses in grape berries. However, changes in resveratrol caused by bud sport in grapes are scarcely reported. In this study, trans-resveratrol and cis-resveratrol were identified and quantified in the grape berries of ‘Summer Black’ and its bud sport ‘Nantaihutezao’ from the veraison to ripening stages using ultra performance liquid chromatography-high resolution tandem mass spectrometry (UPLC-HRMS). We found that bud sport accumulates the trans-resveratrol earlier and increases the contents of cis-resveratrol in the earlier stages but decreases its contents in the later stages. Simultaneously, we used RNA-Seq to identify 51 transcripts involved in the stilbene pathways. In particular, we further identified 124 and 19 transcripts that negatively correlated with the contents of trans-resveratrol and cis-resveratrol, respectively, and four transcripts encoding F3'5'H that positively correlated with the contents of trans-resveratrol by weighted gene co-expression network analysis (WGCNA). These transcripts may play important roles in relation to the synergistic regulation of metabolisms of resveratrol. The results of this study can provide a theoretical basis for the genetic improvement of grapes.

Differences in Anthocyanin Accumulation Patterns and Related Gene Expression in Two Varieties of Red Pear

Red pear is a popular fruit that is appreciated for its attractive and distinctive appearance and mild flavor. In this study, we investigated the mechanism underlying the red coloration of pear skin using the ‘Xinqihong’ cultivar—which was selected as a spontaneous bud sport mutant of the ‘Xinli 7′(Pyrus betulifolia Rehd.) variety and has a stronger red color that is retained in the mature fruit—as an experimental model. We compared the phenotype and gene expression patterns of the two varieties and found no significant differences at the early stage of fruit development. However, although the red color of ‘Xinli 7′ fruits began to fade 107 days after full bloom, that of ‘Xinqihong’ fruits persisted until the time of harvest. Transcriptome sequencing identified 639 genes that were differentially expressed between the two varieties, including genes related to light, calcium, and hormone signaling (e.g., PbPIF3, PbGA2ox, and the calmodulin related genes). Moreover, anthocyanin biosynthesis genes were downregulated as the red color of ‘Xinli 7′ fruits faded. These results provide insight into the molecular basis of color differences in red pears that can be useful for improving its fruit quality and commercial value.

Endogenous Auxin Content Contributes to Larger Size of Apple Fruit

Fruit size is an important economic trait that is controlled by multiple genes. However, the regulatory mechanism for fruit size remains poorly understood. A bud sport variety of “Longfeng” (LF) apple (Malus domestica) was identified and named “Grand Longfeng” (GLF). The fruit size of GLF is larger than that of LF, and both varieties are diploid. We found that the cell size in GLF fruit was larger than that of LF. Then, we compared the fruit transcriptomes of the two varieties using RNA-Seq technology. A total of 1166 differentially expressed genes (DEGs) were detected between GLF and LF fruits. The KEGG analysis revealed that the phytohormone pathway was the most enriched, in which most of the DEGs were related to auxin signaling. Moreover, the endogenous auxin levels of GLF fruit were higher than those of LF. The expressions of auxin synthetic genes, including MdTAR1 and MdYUCCA6, were higher in GLF fruit than LF. Collectively, our findings suggest that auxin plays an important role in fruit size development.

PbGA2ox8 induces vascular-related anthocyanin accumulation and contributes to red stripe formation on pear fruit

Fruit with stripes, which are generally longitudinal, can occur naturally, but the bioprocesses underlying this phenomenon are unclear. Previously, we observed an atypical anthocyanin distribution that caused red-striped fruit on the spontaneous pear bud sport “Red Zaosu” (Pyrus bretschneideri Rehd.). In this study, comparative transcriptome analysis of the sport and wild-type “Zaosu” revealed that this atypical anthocyanin accumulation was tightly correlated with abnormal overexpression of the gene-encoding gibberellin (GA) 2-beta-dioxygenase 8, PbGA2ox8. Consistently, decreased methylation was also observed in the promoter region of PbGA2ox8 from “Red Zaosu” compared with “Zaosu”. Moreover, the GA levels in “Red Zaosu” seedlings were lower than those in “Zaosu” seedlings, and the application of exogenous GA4 reduced abnormal anthocyanin accumulation in “Red Zaosu”. Transient overexpression of PbGA2ox8 reduced the GA4 level and caused anthocyanin accumulation in pear fruit skin. Moreover, the presence of red stripes indicated anthocyanin accumulation in the hypanthial epidermal layer near vascular branches (VBs) in “Red Zaosu”. Transient overexpression of PbGA2ox8 resulting from vacuum infiltration induced anthocyanin accumulation preferentially in calcium-enriched areas near the vascular bundles in pear leaves. We propose a fruit-striping mechanism, in which the abnormal overexpression of PbGA2ox8 in “Red Zaosu” induces the formation of a longitudinal array of anthocyanin stripes near vascular bundles in fruit.

Parallel Bud Mutation Sequencing Reveals that Fruit Sugar and Acid Metabolism Potentially Influence Stress in Malus

Apple sugar and acid are the most important traits of apple fruit. Bud sport cultivars can provide abundant research materials for functional gene studies in apple. In this study, using bud sport materials with a rather different sugar and acid flavor, i.e., “Jonathan” and “Sweet Jonathan”, we profiled the whole genome variations and transcriptional regulatory network during fruit developmental stages using whole genome sequencing and RNA-sequencing. Variation analysis identified 4,198,955 SNPs, 319,494 InDels, and 32,434 SVs between the two cultivars. In total, 4313 differentially expressed genes among all of the d 44,399 genes expressed were identified between the two cultivars during fruit development, and functional analysis revealed stress response and signal transduction related genes were enriched. Using 24,047 genes with a more variable expression value, we constructed 28 co-expression modules by weighted correlation network analysis. Deciphering of 14 co-expression modules associated with sugar or acid accumulation during fruit development revealed the hub genes associated with sugar and acid metabolism, e.g., MdDSP4, MdINVE, and MdSTP7. Furthermore, exploration of the intra network of the co-expression module indicated the close relationship between sugar and acid metabolism or sugar and stress. Motif-based sequence analysis of the 17 differentially expressed ATP-binding cassette transporter genes and Yeast one-hybrid assay identified and confirmed a transcription factor, MdBPC6, regulating the ATP-binding cassette (ABC) transporter genes and potentially participating in the apple fruit development or stress response. Collectively, all of the results demonstrated the use of parallel bud mutation sequencing and identified hub genes, and inferred regulatory relationships providing new information about apple fruit sugar and acid accumulation or stress response.