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Citrus, one of the largest fruit crops with global economic and nutritional importance, contains fruit known as hesperidium with unique morphological types. Citrus fruit ripening is accompanied by chlorophyll degradation and carotenoid biosynthesis, which are indispensably linked to color formation and the external appearance of citrus fruits. However, the transcriptional coordination of these metabolites during citrus fruit ripening remains unknown. Here, we identified the MADS-box transcription factor CsMADS3 in Citrus hesperidium that coordinates chlorophyll and carotenoid pools during fruit ripening. CsMADS3 is a nucleus-localized transcriptional activator, and its expression is induced during fruit development and coloration. Overexpression of CsMADS3 in citrus calli, tomato (Solanum lycopersicum), and citrus fruits enhanced carotenoid biosynthesis and upregulated carotenogenic genes while accelerating chlorophyll degradation and upregulating chlorophyll degradation genes. Conversely, the interference of CsMADS3 expression in citrus calli and fruits inhibited carotenoid biosynthesis and chlorophyll degradation and down-regulated the transcription of related genes. Further assays confirmed that CsMADS3 directly binds and activates the promoters of phytoene synthase 1 (CsPSY1) and chromoplast-specific lycopene β-cyclase (CsLCYb2), two key genes in the carotenoid biosynthetic pathway, and STAY-GREEN (CsSGR), a critical chlorophyll degradation gene, which explained the expression alterations of CsPSY1, CsLCYb2, and CsSGR in the above transgenic lines. These findings reveal the transcriptional coordination of chlorophyll and carotenoid pools in the unique hesperidium of Citrus and may contribute to citrus crop improvement.

As an essential horticultural crop, Citrus has carotenoid diversity, which affects its aesthetic and nutritional values. β,β-Xanthophylls are the primary carotenoids accumulated in citrus fruits, and non-heme di-iron carotene hydroxylase (BCH) enzymes are mainly responsible for β,β-xanthophyll synthesis. Previous studies have focused on the hydroxylation of BCH1, but the role of its paralogous gene in citrus, BCH2, remains largely unknown. In this study, we revealed the β-hydroxylation activity of citrus BCH2 (CsBCH2) for the first time through the functional complementation assay using Escherichia coli, although CsBCH2 exhibited a lower activity in hydroxylating β-carotene into β-cryptoxanthin than citrus BCH1 (CsBCH1). Our results showed that overexpression of CsBCH2 in citrus callus increased xanthophyll proportion and plastoglobule size with feedback regulation of carotenogenic gene expression. This study revealed the distinct expression patterns and functional characteristics of two paralogous genes, CsBCH1 and CsBCH2, and illustrated the backup compensatory role of CsBCH2 for CsBCH1 in citrus xanthophyll biosynthesis. The independent function of CsBCH2 and its cooperative function with CsBCH1 in β-cryptoxanthin biosynthesis suggested the potential of CsBCH2 to be employed for expanding the synthetic biology toolkit in carotenoid engineering.

To measure stomatal traits automatically and nondestructively, a new method for detecting stomata and extracting stomatal traits was proposed. Two portable microscopes with different resolutions (TipScope with a 40× lens attached to a smartphone and ProScope HR2 with a 400× lens) are used to acquire images of living stomata in maize leaves. FPN model was used to detect stomata in the TipScope images and measure the stomata number and stomatal density. Faster RCNN model was used to detect opening and closing stomata in the ProScope HR2 images, and the number of opening and closing stomata was measured. An improved CV model was used to segment pores of opening stomata, and a total of 6 pore traits were measured. Compared to manual measurements, the square of the correlation coefficient (R2 ) of the 6 pore traits was higher than 0.85, and the mean absolute percentage error (MAPE) of these traits was 0.02%-6.34%. The dynamic stomata changes between wild-type B73 and mutant Zmfab1a were explored under drought and re-watering condition. The results showed that Zmfab1a had a higher resilience than B73 on leaf stomata. In addition, the proposed method was tested to measure the leaf stomatal traits of other nine species. In conclusion, a portable and low-cost stomata phenotyping method that could accurately and dynamically measure the characteristic parameters of living stomata was developed. An open-access and user-friendly web portal was also developed which has the potential to be used in the stomata phenotyping of large populations in the future.

Senescence is a gradual physiological process involving the integration of numerous internal and environmental signals. Abscisic acid (ABA) is a well-known inducer of senescence. However, the regulatory mechanisms underlying ABA-mediated senescence remain largely unknown. Here, we report that the citrus homeodomain leucine zipper I (HD-ZIP I) transcription factor CsHB5 functions as a regulator of ABA-triggered senescence. CsHB5 acts as a nucleus-localized transcriptional activator, the expression of which appeared to be closely associated with citrus senescence. Overexpression of CsHB5 in citrus calli upregulated the expression of ABA- and reactive oxygen species (ROS)-related genes, and significantly increased the content of ABA and hydrogen peroxide (H2 O2 ), whereas silencing CsHB5 in citrus calli downregulated the expression of ABA-related genes. Additionally, heterogenous overexpression of CsHB5 in Solanum lycopersicum (tomato) and Arabidopsis thaliana (Arabidopsis) leads to early leaf yellowing under dark-induced senescence conditions. Meanwhile, the levels of ABA and H2 O2 in transgenic tomatoes increased significantly and the lycopene content decreased. Transcriptome analysis of CsHB5-overexpressing citrus calli and tomato showed that CsHB5 was involved in multiple senescence-associated processes, including chlorophyll degradation, nutrient compound biosynthesis and transport, as well as ABA and ROS signal transduction. The results of yeast one-hybrid assays, electrophoretic mobility shift assays and dual luciferase assays indicated that CsHB5 directly binds to the promoters of ABA biosynthetic genes, including β-carotene hydroxylase 1 (BCH1) and 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), thereby activating their transcription. Our findings revealed that CsHB5 participates in senescence, at least partly, by directly controlling ABA accumulation. Our work provides insight into the regulatory mechanisms underlying ABA-mediated senescence.

Chromoplast-specific lycopene β-cyclase (LCYb2) is a critical carotenogenic enzyme, which controls the massive accumulation of downstream carotenoids, especially provitamin A carotenoids, in citrus. Its regulatory metabolism is largely unknown. Here, we identified a group I ethylene response factor, CsERF061, in citrus by yeast one-hybrid screen with the promoter of LCYb2. The expression of CsERF061 was induced by ethylene. Transcript and protein levels of CsERF061 were increased during fruit development and coloration. CsERF061 is a nucleus-localized transcriptional activator, which directly binds to the promoter of LCYb2 and activates its expression. Overexpression of CsERF061 in citrus calli and tomato fruits enhanced carotenoid accumulation by increasing the expression of key carotenoid pathway genes, and increased the number of chromoplasts needed to sequester the elevated concentrations of carotenoids, which was accompanied by changes in the concentrations of abscisic acid and gibberellin. Electrophoretic mobility shift and dual-luciferase assays verified that CsERF061 activates the promoters of nine other key carotenoid pathway genes, PSY1, PDS, CRTISO, LCYb1, BCH, ZEP, NCED3, CCD1, and CCD4, revealing the multitargeted regulation of CsERF061. Collectively, our findings decipher a novel regulatory network of carotenoid enhancement by CsERF061, induced by ethylene, which will be useful for manipulating carotenoid accumulation in citrus and other plants.

Carotenoids provide precursors for the biosynthesis of strigolactones, which are a new class of hormones that are essential in phosphate (Pi) signaling during plant development. Carotenoid metabolism is a finely tuned pathway, but our understanding of the regulation mechanisms is still limited. In this study, we isolated a protein designated as CsPHL3 from citrus. CsPHL3 belonged to the Pi starvation response factor (PHR)-like subclade and was upregulated by low Pi. Acting as a nucleus-localized protein with transactivation activity, CsPHL3 bound directly to activate the promoter of a key metabolic gene, lycopene β-cyclase1 (LCYb1). Transgenic analysis revealed that the CsPHL3-overexpressing tomato plants exhibited abnormal growth, like the plants grew under limited Pi conditions. The transgenic lines showed reduced carotenoid contents and elevated expression of LCYb genes but downregulation of other key carotenogenic genes, including phytoene synthase (PSY). Moreover, CsPHL3 induced anthocyanin biosynthesis and affected Pi signaling in the transgenic plants. We further demonstrated that the expression of PSY was negatively regulated by CsPHL3 and high Pi. It is concluded that CsPHL3 is a Pi starvation response factor that negatively regulates carotenoid metabolism by modulating the expression of carotenogenic genes. Establishment of the CsPHL3-CsLCYb1 network provides new valuable knowledge of the function and underlying mechanism of PHR transcription factors and expands our understanding of the complex regulation mechanisms of carotenoid biosynthesis.

High-throughput phenotyping of yield-related traits is meaningful and necessary for rice breeding and genetic study. The conventional method for rice yield-related trait evaluation faces the problems of rice threshing difficulties, measurement process complexity, and low efficiency. To solve these problems, a novel intelligent system, which includes an integrated threshing unit, grain conveyor-imaging units, threshed panicle conveyor-imaging unit, and specialized image analysis software has been proposed to achieve rice yield trait evaluation with high throughput and high accuracy. To improve the threshed panicle detection accuracy, the Region of Interest Align, Convolution Batch normalization activation with Leaky Relu module, Squeeze-and-Excitation unit, and optimal anchor size have been adopted to optimize the Faster-RCNN architecture, termed ‘TPanicle-RCNN,’ and the new model achieved F1 score 0.929 with an increase of 0.044, which was robust to indica and japonica varieties. Additionally, AI cloud computing was adopted, which dramatically reduced the system cost and improved flexibility. To evaluate the system accuracy and efficiency, 504 panicle samples were tested, and the total spikelet measurement error decreased from 11.44 to 2.99% with threshed panicle compensation. The average measuring efficiency was approximately 40 s per sample, which was approximately twenty times more efficient than manual measurement. In this study, an automatic and intelligent system for rice yield-related trait evaluation was developed, which would provide an efficient and reliable tool for rice breeding and genetic research.

Carotenoids are indispensable for both human health and plant survival. Citrus, is one of the fruit crops richest in carotenoid compounds, with approximately 115 kinds of carotenoids; tremendous diversity in carotenoids composition and concentration exists among various species, showing different colors from nearly white to crimson. The carotenoid biosynthetic pathway and the key carotenogenic genes have been identified in citrus; however, the underlying regulatory mechanisms remain unclear. In this study, among the main species of genus Citrus (primitive, wild, and cultivated), we detected carotenoids in flavedo using High-Performance Liquid Chromatography, and analyzed variations in cis-acting elements in the promoters of key carotenoid pathway genes. Intriguingly, both carotenoid composition and content were generally increased during the evolution of citrus, and the corresponding variations in the promoters were identified, including the gain or loss of critical environmental stress-responsive elements and hormone-responsive elements, which are closely associated with carotenoid enhancement. In addition, pummelo has the most heat-responsive elements, but the Mangshan mandarin does not have this element in the promoters of PSY, which is highly related to their geographical origin and indicate that temperature is a critical environmental signal influencing carotenoid accumulation. Moreover, the abscisic acid-responsive motif was rich in almost all the seven species, but the ethylene-responsive motif was deficient, which demystified the unique phytohormone regulation mechanism of carotenoid accumulation in citrus. Overall, our study provides new insights into the molecular regulatory mechanism of carotenoid enhancement in the evolution of citrus, which can facilitate breeding and cultivation efforts to improve the nutritional quality and esthetic value in citrus and hopefully other fruit crops.

Self-incompatibility (SI) is an important mechanism that prevents self-fertilization and inbreeding in flowering plants. The most widespread SI system utilizes S ribonucleases (S-RNases) and S-locus F-boxes (SLFs) as S determinants. In citrus, SI is ancestral, and Citrus maxima (pummelo) is self-incompatible, while Citrus reticulata (mandarin) and its hybrids are self-compatible (SC). Here, we identify nine highly polymorphic pistil-specific, developmentally expressed S-RNases from pummelo that segregate with S haplotypes in a gametophytic manner and cluster with authentic S-RNases. We provide evidence that these S-RNases function as the female S determinants in citrus. Moreover, we show that each S-RNase is linked to approximately nine SLFs. In an analysis of 117 citrus SLF and SFL-like (SLFL) genes, we reveal that they cluster into 12 types and that the S-RNases and intra-haplotypic SLF and SLFL genes co-evolved. Our data support the notion that citrus have a S locus comprising a S-RNase and several SLFs that fit the non-self-recognition model. We identify a predominant single nucleotide mutation, Sm-RNase, in SC citrus, which provides a 'natural' loss of function. We show that SI-SC transitions due to the Sm-RNase initially arose in mandarin, spreading to its hybrids and became fixed. Identification of an evolutionarily distant new genus utilizing the S-RNase-based SI system, >100 million years separated from the nearest S-RNase family, is a milestone for evolutionary comparative studies.

A low-cost portable wild phenotyping system is useful for breeders to obtain detailed phenotypic characterization to identify promising wild species. However, compared with the larger, faster, and more advanced in-laboratory phenotyping systems developed in recent years, the progress for smaller phenotyping systems, which provide fast deployment and potential for wide usage in rural and wild areas, is quite limited. In this study, we developed a portable whole-plant on-device phenotyping smartphone application running on Android that can measure up to 45 traits, including 15 plant traits, 25 leaf traits and 5 stem traits, based on images. To avoid the influence of outdoor environments, we trained a DeepLabV3+ model for segmentation. In addition, an angle calibration algorithm was also designed to reduce the error introduced by the different imaging angles. The average execution time for the analysis of a 20-million-pixel image is within 2,500 ms. The application is a portable on-device fast phenotyping platform providing methods for real-time trait measurement, which will facilitate maize phenotyping in field and benefit crop breeding in future.

Self-incompatibility (SI) substantially restricts the yield and quality of citrus. Therefore, breeding and analyzing self-compatible germplasm is of great theoretical and practical significance for citrus. Here, we focus on the mechanism of a self-compatibility mutation in ‘Guiyou No. 1’ pummelo (Citrus maxima), which is a spontaneous mutant of ‘Shatian’ pummelo (Citrus maxima, self-incompatibility). The rate of fruit set and the growth of pollen tubes in the pistil confirmed that a spontaneous mutation in the pistil is responsible for the self-compatibility of ‘Guiyou No. 1’. Segregation ratios of the S genotype in F1 progeny, expression analysis, and western blotting validated that the reduced levels of S2-RNase mRNA contribute to the loss of SI in ‘Guiyou No. 1’. Furthermore, we report a phased assembly of the ‘Guiyou No. 1’ pummelo genome and obtained two complete and well-annotated S haplotypes. Coupled with an analysis of SV variations, methylation levels, and gene expression, we identified a candidate gene (CgHB40), that may influence the regulation of the S2-RNase promoter. Our data provide evidence that a mutation that affects the pistil led to the loss of SI in ‘Guiyou No. 1’ by influencing a poorly understood mechanism that affects transcriptional regulation. This work significantly advances our understanding of the genetic basis of the SI system in citrus and provides information on the regulation of S-RNase genes.

In citrus, PH4–Noemi, a regulatory complex, controls proanthocyanidin accumulation by activating the transcription of proanthocyanidin biosynthetic genes, and also regulates expression of Noemi, promoting proanthocyanidin accumulation by positive feedback., Proanthocyanidins (PAs; or condensed tannins) are a major class of flavonoids that contribute to citrus fruit quality. However, the molecular mechanism responsible for PA biosynthesis and accumulation in citrus remains unclear. Here, we identify a PH4–Noemi regulatory complex that regulates proanthocyanidin biosynthesis in citrus. Overexpression of PH4 or Noemi in citrus calli activated the expression of PA biosynthetic genes and significantly increased the PA content. Interestingly, Noemi was also shown to be up-regulated in CsPH4-overexpressing lines compared with wild-type calli. Simultaneously, CsPH4 partially complemented the PA-deficient phenotype of the Arabidopsis tt2 mutant and promoted PA accumulation in the wild-type. Further analysis revealed that CsPH4 interacted with Noemi, and together these proteins synergistically activated the expression of PA biosynthetic genes by directly binding to the MYB-recognizing elements (MRE) of the promoters of these genes. Moreover, CsPH4 could directly bind to the promoter of Noemi and up-regulate the expression of this gene. These findings explain how the CsPH4–Noemi regulatory complex contributes to the activation of PA biosynthetic genes via a positive feedback loop and provide new insights into the molecular mechanisms underlying PA biosynthesis, which can be effectively employed for metabolic engineering to improve citrus fruit quality.

Summary Hongkong kumquat (Fortunella hindsii) is a wild citrus species characterized by dwarf plant height and early flowering. Here, we identified the monoembryonic F. hindsii (designated as ‘Mini‐Citrus’) for the first time and constructed its selfing lines. This germplasm constitutes an ideal model for the genetic and functional genomics studies of citrus, which have been severely hindered by the long juvenility and inherent apomixes of citrus. F. hindsii showed a very short juvenile period (~8 months) and stable monoembryonic phenotype under cultivation. We report the first de novo assembled 373.6 Mb genome sequences (Contig‐N50 2.2 Mb and Scaffold‐N50 5.2 Mb) for F. hindsii. In total, 32 257 protein‐coding genes were annotated, 96.9% of which had homologues in other eight Citrinae species. The phylogenomic analysis revealed a close relationship of F. hindsii with cultivated citrus varieties, especially with mandarin. Furthermore, the CRISPR/Cas9 system was demonstrated to be an efficient strategy to generate target mutagenesis on F. hindsii. The modifications of target genes in the CRISPR‐modified F. hindsii were predominantly 1‐bp insertions or small deletions. This genetic transformation system based on F. hindsii could shorten the whole process from explant to T1 mutant to about 15 months. Overall, due to its short juvenility, monoembryony, close genetic background to cultivated citrus and applicability of CRISPR, F. hindsii shows unprecedented potentials to be used as a model species for citrus research.

Deciphering the genetic basis of plant secondary metabolism will provide useful insights for genetic improvement and enhance our fundamental understanding of plant biological processes. Although citrus plants are among the most important fruit crops worldwide, the genetic basis of secondary metabolism in these plants is largely unknown. Here, we use a high-density linkage map to dissect large-scale flavonoid metabolic traits measured in different tissues (young leaf, old leaf, mature pericarp, and mature pulp) of an F1 pseudo-testcross citrus population. We detected 80 flavonoids in this population and identified 138 quantitative trait loci (QTLs) for 57 flavonoids in these four tissues. Based on transcriptional profiling and functional annotation, twenty-one candidate genes were identified, and one gene encoding flavanone 3-hydroxylase (F3H) was functionally verified to result in naturally occurring variation in dihydrokaempferol content through genetic variations in its promoter and coding regions. The abundant data resources collected for diverse citrus germplasms here lay the foundation for complete characterization of the citrus flavonoid biosynthetic pathway and will thereby promote efficient utilization of metabolites in citrus quality improvement.

In this study, based on automatic control and image processing, a high-throughput and low-cost maize ear traits scorer (METS) was developed for the automatic measurement of 34 maize ear traits. In total, 813 maize ears were measured using METS, and the results showed that the square of the correlation coefficient (R(2)) of the manual measurements versus the automatic measurements for ear length, ear diameter, and kernel thickness were 0.96, 0.79, and 0.85, respectively. These maize ear traits could be used to classify the type, and the results showed that the classification accuracy of the support vector machine (SVM) model for the test set was better than that of the random forest (RF) model. In addition, the general applicability of the image analysis pipeline was also demonstrated on other independent maize ear phenotyping platforms. In conclusion, equipped with image processing and automatic control technologies, we have developed a high-throughput method for maize ear scoring, which could be popularized in maize functional genetics, genomics, and breeding applications. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-021-01205-4.

Domesticated citrus varieties are woody perennials and interspecific hybrid crops of global economic and nutritional importance. The citrus fruit “hesperidium” is a unique morphological innovation not found in any other plant lineage. Efforts to improve the nutritional quality of the fruit are predicated on understanding the underlying regulatory mechanisms responsible for fruit development, including temporal control of chlorophyll degradation and carotenoid biosynthesis. Here, we investigated the molecular basis of the navel orange (Citrus sinensis) brown flavedo mutation, which conditions flavedo that is brown instead of orange. To overcome the limitations of using traditional genetic approaches in citrus and other woody perennials, we developed a strategy to elucidate the underlying genetic lesion. We used a multi-omics approach to collect data from several genetic sources and plant chimeras to successfully decipher this mutation. The multi-omics strategy applied here will be valuable in driving future gene discovery efforts in citrus as well as in other woody perennial plants. The comparison of transcriptomic and genomic data from multiple genotypes and plant sectors revealed an underlying lesion in the gene encoding STAY-GREEN (SGR) protein, which simultaneously regulates carotenoid biosynthesis and chlorophyll degradation. However, unlike SGR of other plant species, we found that the carotenoid and chlorophyll regulatory activities could be uncoupled in the case of certain SGR alleles in citrus and thus we propose a model for the molecular mechanism underlying the brown flavedo phenotype. The economic and nutritional value of citrus makes these findings of wide interest. The strategy implemented, and the results obtained, constitute an advance for agro-industry by driving opportunities for citrus crop improvement.

Carotenoids in citrus contribute to the quality of the fruit, but the mechanism of its transcriptional regulation is fairly unknown. Here, we characterized a citrus FRUITFULL sub-clade MADS gene, CsMADS5, that was ripening-inducible and acted as a nucleus-localized trans-activator. Transient overexpression of CsMADS5 in citrus induced fruit coloration and enhanced carotenoid concentrations. The expression of carotenogenic genes including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene β-cyclase 1 (LCYb1) was increased in the peels of fruits overexpressing CsMADS5. Similar results were observed from stable overexpression of CsMADS5 in tomato fruits and citrus calli, even though the effect of CsMADS5 on carotenoid metabolism in transgenic citrus calli was limited. Further biochemical analyses demonstrated that CsMADS5 activated the transcription of PSY, PDS, and LCYb1 by directly binding to their promoters. We concluded that CsMADS5 positively regulates carotenoid biosynthesis in fruits by directly activating the transcription of carotenogenic genes. Moreover, CsMADS5 physically interacted with a positive regulator CsMADS6, indicating that CsMADS5 may form an enhancer complex with CsMADS6 to synergistically promote carotenoid accumulation. These findings expand our understanding of the complex transcriptional regulatory hierarchy of carotenoid biosynthesis during fruit ripening.

Combining high-throughput micro-CT-RGB phenotyping with genome-wide association study reveals the genetic architecture of tiller development in the indica subpopulation of rice., Manual phenotyping of rice tillers is time consuming and labor intensive, and lags behind the rapid development of rice functional genomics. Thus, automated, non-destructive methods of phenotyping rice tiller traits at a high spatial resolution and high throughput for large-scale assessment of rice accessions are urgently needed. In this study, we developed a high-throughput micro-CT-RGB imaging system to non-destructively extract 739 traits from 234 rice accessions at nine time points. We could explain 30% of the grain yield variance from two tiller traits assessed in the early growth stages. A total of 402 significantly associated loci were identified by genome-wide association study, and dynamic and static genetic components were found across the nine time points. A major locus associated with tiller angle was detected at time point 9, which contained a major gene, TAC1. Significant variants associated with tiller angle were enriched in the 3ʹ-untranslated region of TAC1. Three haplotypes for the gene were found, and rice accessions containing haplotype H3 displayed much smaller tiller angles. Further, we found two loci containing associations with both vigor-related traits identified by high-throughput micro-CT-RGB imaging and yield. The superior alleles would be beneficial for breeding for high yield and dense planting.

A novel NAC transcription factor, CrNAC036, can interact with CrMYB68 to synergistically regulate the ripening process of citrus fruit by down-regulating the expression of 9-cis-epoxycarotenoid dioxygenase 5., Although abscisic acid (ABA) is a vital regulator of fruit ripening and several transcription factors have been reported to regulate ABA biosynthesis, reports of the effect of ABA on citrus ripening and the regulation of its biosynthesis by a multiple-transcription-factor complex are scarce. In the present study, a systematic metabolic, cytological, and transcriptome analysis of an ABA-deficient mutant (MT) of Citrus reticulata cv. Suavissima confirmed the positive effect of ABA on the citrus ripening process. The analysis of transcriptome profiles indicated that CrNAC036 played an important role in the ABA deficiency of the mutant, most likely due to an effect on the expression of 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5). Electrophoretic mobility shift assays and dual luciferase assays demonstrated that CrNAC036 can directly bind and negatively regulate CrNCED5 expression. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and dual luciferase assays demonstrated that CrNAC036 interacted with CrMYB68, also down-regulating the expression of CrNCED5. Taken together, our results suggest that CrNAC036 and CrMYB68 synergistically inhibit ABA biosynthesis in citrus fruit by regulating the expression of CrNCED5.

This work reports the development of suspension culture system of transgenic Marsh grapefruit (Citrus paradisi Macf., Rutaceae) callus overexpressing bacterial phytoene synthase; and the use of this suspension culture to investigate the effects of β-cyclocitral on carotenoid content and composition. At a β-cyclocitral concentration of 0.5 mM and after ten days cultivation, analysis of the carotenoids showed a significant increase in the content of β-, α-carotene, and phytoene predominantly. The maximal increase in total provitamin A carotenoids content following β-cyclocitral application was ~2-fold higher than the control, reaching 245.8 μg/g DW. The trend for increased transcript levels of biosynthetic genes PSY and ZDS correlated with the enhancement of the content of these carotenes following β-cyclocitral treatment and GC-MS based metabolite profiling showed significant changes of metabolite levels across intermediary metabolism. These findings suggest that β-cyclocitral can act as a chemical elicitor, to enhance the formation of carotenes in citrus suspension-cultured cells (SCC), which could be utilized in studying the regulation of carotenoid biosynthesis and biotechnological application to the renewable production of nutritional carotenoids.

Peach gummosis, caused by Lasiodiplodia theobromae, is one of the most prevalent diseases that affects peach production. In this study, we investigated the effect of zinc sulfate on inoculated peach shoots, as well as on the growth, morphology, and pathogenicity of L. theobromae in vitro, in the laboratory. Zinc deficiency was detected in diseased peach shoots by micronutrient analysis (Cu, Mn, and Zn) and confirmed by the measurement of transcript levels of zinc transporters (ZIP4, HAM4, and ZAT). The zinc was transferred from the diseased peach shoots to the peach gum. Applying zinc sulfate to the diseased peach shoots reduced the severity of peach gummosis, showing significantly reduced lesion size and gum weight, as well as downregulation of cell wall degradation-related gene (PG and PME) compared with the control. Zinc sulfate also specifically controlled peach gummosis under L. theobromae phytotoxin stress and induced the expression of defense-related genes (PR4, CHI, PAL, PGIP, and GNS3). In addition, in vitro mycelial growth of L. theobromae was significantly inhibited by zinc sulfate compared with the control. Zinc sulfate caused abnormal hyphae at 25 mM and swelling hyphal tips at 50 mM. Exposure of L. theobromae to zinc sulfate for 20 min inhibited the ability of the pathogen to cause peach gummosis. Our physiological and molecular data demonstrated that zinc sulfate has a dual function by reducing susceptibility in the host and by direct inhibition of the pathogen.

Carotenoids and apocarotenoids act as phytohormones and volatile precursors that influence plant development and confer aesthetic and nutritional value critical to consumer preference. Citrus fruits display considerable natural variation in carotenoid and apocarotenoid pigments. In this study, using an integrated genetic approach we revealed that a 5' cis-regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C30 apocarotenoids responsible for red coloration of citrus peel. Functional analyses demonstrated that in addition the known role in synthesizing β-citraurin, CCD4b is also responsible for the production of another important C30 apocarotenoid pigment, β-citraurinene. Furthermore, analyses of the CCD4b promoter and transcripts from various citrus germplasm accessions established a tight correlation between the presence of a putative 5' cis-regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C30 apocarotenoid-rich red-peeled accessions. Phylogenetic analysis provided further evidence that functional diversification of CCD4b and naturally occurring variation of the CCD4b promoter resulted in the stepwise evolution of red peels in mandarins and their hybrids. Taken together, our findings provide new insights into the genetic and evolutionary basis of apocarotenoid diversity in plants, and would facilitate breeding efforts that aim to improve the nutritional and aesthetic value of citrus and perhaps other fruit crops.

Elaioplasts of citrus peel are colorless plastids which accumulate significant amounts of terpenes. However, other functions of elaioplasts have not been fully characterized to date. Here, a LC–MS/MS shotgun technology was applied to identify the proteins from elaioplasts that were highly purified from young fruit peel of kumquat. A total of 655 putative plastid proteins were identified from elaioplasts according to sequence homology in silico and manual curation. Based on functional classification via Mapman, ~50% of the identified proteins fall into six categories, including protein metabolism, transport, and lipid metabolism. Of note, elaioplasts contained ATP synthase and ADP, ATP carrier proteins at high abundance, indicating important roles for ATP generation and transport in elaioplast biogenesis. Additionally, a comparison of proteins between citrus chromoplast and elaioplast proteomes suggest a high level of functional conservation. However, some distinctive protein profiles were also observed in both types of plastids notably for isoprene biosynthesis in elaioplasts, and carotenoid metabolism in chromoplasts. In conclusion, this comprehensive proteomic study provides new insights into the major metabolic pathways and unique characteristics of elaioplasts and chromoplasts in citrus fruit., Citrus fruits: The proteins of elaioplasts A study of the proteins in bodies called elaioplasts in citrus fruit cells will help to clarify elaioplast formation and functions. Plant cells contain several types of membrane-bound bodies called plastids, with elaioplasts being an incompletely characterized type. They are known to store oils in the cells, especially molecules called terpenes that influence the fragrances and flavors of citrus fruits. Researchers in China, led by Yunliu Zeng at Huazhong Agricultural University, analyzed the protein molecules in elaioplasts of kumquat peel to investigate possible roles for elaioplasts other than oil storage. They identified more than 600 proteins and assigned likely functions to many. The results suggest the proteins are involved in chemical energy transactions and the manufacture of various categories of biomolecules. This will guide research to fully characterize the activities and significance of elaioplasts.

Peach gummosis, caused by Botryosphaeria spp. fungi, is the process of gum accumulation and exudation in plants. Ethephon (2-chloroethylphosphonic acid) has profound effects on plants, including enhanced production of secondary metabolites and regulation of plant diseases. This study investigates the effects of application of ethephon before and after inoculation with Lasiodiplodia theobromae on gum formation. Gum formation was promoted by ethephon treatment prior to pathogen inoculation, but inhibited by ethephon applied after the pathogen. The inhibitory effect was counteracted by 1-methylcyclopropane, which is an ethylene signal inhibitor. 1-methylcyclopropane also promoted gum formation. Exposure of three isolates of Botryosphaeria to ethephon inhibited mycelial growth. Both treatment methods increased the sugar content at 12 and 24 h post-inoculation (hpi). However, the sucrose, glucose and fructose contents were significantly higher in shoots with ethephon post-treatment (application of ethephon after the pathogen inoculation) than those in shoots with ethephon pre-treatment (application of ethephon prior to pathogen inoculation) at 48 and 72 hpi. The expression of two putative senescence-related genes, SEN2 and SEN4, were significantly enhanced in pre- and post-treated shoots with ethephon at 24, 48 and 72 hpi. Ethephon application also up-regulated expression of the pathogenesis-related protein PR4 while down-regulating PR1a and PR10. The results show that ethephon has a dual function in regulating gum formation by affecting both the peach shoots and the pathogen.

Peach gummosis is a disease caused by Lasiodiplodia theobromae and a major problem in peach production. The disease is characterized by gum exudation from fungal infection sites on tree trunks, branches, and fruits. In this study, we found that high humidity and mechanical wounds promote gummosis development, whereas different light intensities and temperature do not affect gummosis after infecting detached current year shoots of the susceptible peach cultivar ‘Spring snow’ with L. theobromae in a laboratory. The chlorophyll a and b contents of the infected shoots were significantly lower than those of the uninfected control shoots, but those of H2O2 and malondialdehyde were increased in the infected tissues. Anthocyanin content and phenylalanine ammonia lyase (PAL) activity were significantly higher in the infected shoots at 1 day after inoculation, which is consistent with significant increases in the transcript levels of genes encoding enzymes involved anthocyanin biosynthesis, such as PAL, chalcone synthase, and flavanone 3-hydroxylase. The transcript levels were induced for defence-related genes, namely, chitinase, beta-1,3-glucanase, polygalacturonase-inhibiting protein, and pathogenesis-related protein 4, but down-regulated for pathogenesis-related protein 10. Cell walls were seriously degraded in response to L. theobromae infection, as observed under transmission electron microscopy. Accordingly, the transcript levels of genes that encode cell wall–degrading enzymes, such as beta-galactosidase, pectin methylesterase 1, and endo-polygalacturonase, were significantly increased in infected peach shoots.

Lasiodiplodia theobromae is a causal agent of peach (Prunus persica L.) tree gummosis, a serious disease affecting peach cultivation and production. However, the molecular mechanism underlying the pathogenesis remains unclear. RNA-Seq was performed to investigate gene expression in peach shoots inoculated or mock-inoculated with L. theobromae. A total of 20772 genes were detected in eight samples; 4231, 3750, 3453, and 3612 differentially expressed genes were identified at 12, 24, 48, and 60 h after inoculation, respectively. Furthermore, 920 differentially co-expressed genes (515 upregulated and 405 downregulated) were found, respectively. Gene ontology annotation revealed that phenylpropanoid biosynthesis and metabolism, uridine diphosphate-glucosyltransferase activity, and photosynthesis were the most differentially regulated processes during gummosis development. Significant differences were also found in the expression of genes involved in glycometabolism and in ethylene and jasmonic acid biosynthesis and signaling. These data illustrate the dynamic changes in gene expression in the inoculated peach shoots at the transcriptome level. Overall, gene expression in defense response and glycometabolism might result in the gummosis of peach trees induced by L. theobromae.

Low temperature storage is widely applied to maintain citrus postharvest fruit quality. In this study, the transcriptional and metabolic changes in the pulp tissue of Citrus reticulata Blanco cv. "Ponkan" were studied for three successive months under cold storage by Affymetrix Citrus GeneChip and gas chromatography, respectively. As many as 2 161 differentially expressed transcripts were identified based on the bayesian hierarchical model. The statistical analysis of gene ontology revealed that defense/stress-related genes were induced quickly, while autophagy-related genes were overrepresented in the late sampling stages, suggesting that the functional shift may coincide with the subsequent steps of chilling development. We further classified the potential regulatory components and concluded that ethylene may play the crucial role in chilling development in this non-climacteric fruit. To cope with complex events, 53 upregulated transcription factors represented regulatory candidates. Within these, the AP2-EREBP, C2H2 and AS2 gene family were overrepresented. Cold storage also causes alterations in various metabolic pathways; a keen interest is paid in deciphering expression changes of soluble sugar genes as increased evidence that soluble sugars act as both osmolytes and metabolite signal molecules. Our results will likely facilitate further studies in this field with promising genetic candidates during chilling.

A citrus NAM, ATAF, and CUC (NAC)-like gene (CitNAC) was isolated from fruit tissues of Citrus sinensis Osbeck using complementary DNA (cDNA) amplified fragment length polymorphism and rapid amplification of cDNA ends techniques. Its full length was 988 bp in which 781 bp form the open reading frame, coding for a protein of 264 amino acids. Sequence comparison revealed that CitNAC possesses the general structural features at the N terminus of the NAC domains. Phylogenetic analysis results showed that CitNAC was closely related to AtNAP and PeNAP, which are involved in plant organ senescence. Gene expression analysis showed that the messenger RNA level of CitNAC was just detected in fruit peel and pulp during fruit ripening or senescence stage. The observed expression pattern of CitNAC along with the result of phylogenetic analysis suggested that CitNAC is related to fruit development and senescence.

Genetic manipulation of carotenoid biosynthesis has become a recent focus for the alleviation of vitamin A deficiency. However, the genetically modified phenotypes often challenge the expectation, suggesting the incomplete comprehension of carotenogenesis. Here, embryogenic calli were engineered from four citrus genotypes as engineered cell models (ECMs) by over-expressing a bacterial phytoene synthase gene (CrtB). Ripe flavedos (the coloured outer layer of citrus fruits), which exhibit diverse natural carotenoid patterns, were offered as a comparative system to the ECMs. In the ECMs, carotenoid patterns showed diversity depending on the genotypes and produced additional carotenoids, such as lycopene, that were absent from the wild-type lines. Especially in the ECMs from dark-grown culture, there emerged a favoured β,β-pathway characterized by a striking accumulation of β-carotene, which was dramatically different from those in the wild-type calli and ripe flavedos. Unlike flavedos that contained a typical chromoplast development, the ECMs sequestered most carotenoids in the amyloplasts in crystal form, which led the amyloplast morphology to show a chromoplast-like profile. Transcriptional analysis revealed a markedly flavedo-specific expression of the β-carotene hydroxylase gene (HYD), which was suppressed in the calli. Co-expression of CrtB and HYD in the ECMs confirmed that HYD predominantly mediated the preferred carotenoid patterns between the ECMs and flavedos, and also revealed that the carotenoid crystals in the ECMs were mainly composed of β-carotene. In addition, a model is proposed to interpret the common appearance of a favoured β,β-pathway and the likelihood of carotenoid degradation potentially mediated by photo-oxidation and vacuolar phagocytosis in the ECMs is discussed.

Expressed sequence tag (EST) analysis of the pulp of the red-fleshed mutant ‘Cara Cara’ navel orange provided a starting point for gene discovery and transcriptome survey during citrus fruit maturation. Interpretation of the EST datasets revealed that the mutant pulp transcriptome held a high section of stress responses related genes, such as the type III metallothionein-like gene (6.0%), heat shock protein (2.8%), Cu/Zn superoxide dismutase (0.8%), late embryogenesis abundant protein 5 (0.8%), etc. 133 transcripts were detected to be differentially expressed between the red mutant and its orange-color wild genotype ‘Washington’ via digital expression analysis. Among them, genes involved in metabolism, defense/stress and signal transduction were statistical overrepresented. Fifteen transcription factors, composed of NAM, ATAF, and CUC transcription factor (NAC); myeloblastosis (MYB); myelocytomatosis (MYC); basic helix-loop-helix (bHLH); basic leucine zipper (bZIP) domain members, were also included. The data reflected the distinct expression profile and the unique regulatory module associated with these two genotypes. Eight differently expressed genes analyzed in digital were validated by quantitative real-time polymerase chain reaction. For structural polymorphism, both simple sequence repeats and single nucleotide polymorphisms (SNP) loci were surveyed; dinucleotide presentation revealed a bias toward AG/GA/TC/CT repeats (52.5%), against GC/CG repeats (0%). SNPs analysis found that transitions (73%) outnumbered transversions (27%). Seventeen potential cultivar-specific and 387 heterozygous SNP loci were detected from ‘Cara Cara’ and ‘Washington’ EST pool.