Comprehensive Profiling of Alternative Splicing and Alternative Polyadenylation during Fruit Ripening in Watermelon (Citrullus lanatus).

Fruit ripening is a highly complicated process that is accompanied by the formation of fruit quality. In recent years, a series of studies have demonstrated post-transcriptional control play important roles in fruit ripening and fruit quality formation. Till now, the post-transcriptional mechanisms for watermelon fruit ripening have not been comprehensively studied. In this study, we conducted PacBio single-molecule long-read sequencing to identify genome-wide alternative splicing (AS), alternative polyadenylation (APA) and long non-coding RNAs (lncRNAs) in watermelon fruit. In total, 6,921,295 error-corrected and mapped full-length non-chimeric (FLNC) reads were obtained. Notably, more than 42,285 distinct splicing isoforms were derived from 5,891,183 intron-containing full-length FLNC reads, including a large number of AS events associated with fruit ripening. In addition, we characterized 21,506 polyadenylation sites from 11,611 genes, 8703 of which have APA sites. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that fructose and mannose metabolism, starch and sucrose metabolism and carotenoid biosynthesis were both enriched in genes undergoing AS and APA. These results suggest that post-transcriptional regulation might potentially have a key role in regulation of fruit ripening in watermelon. Taken together, our comprehensive PacBio long-read sequencing results offer a valuable resource for watermelon research, and provide new insights into the molecular mechanisms underlying the complex regulatory networks of watermelon fruit ripening.

An update on sugar allocation and accumulation in fruits.

Fruit sweetness is determined by the amount and composition of sugars in the edible flesh. The accumulation of sugar is a highly orchestrated process that requires coordination of numerous metabolic enzymes and sugar transporters. This coordination enables partitioning and long-distance translocation of photoassimilates from source tissues to sink organs. In fruit crops, sugars ultimately accumulate in the sink fruit. Whereas tremendous progress has been achieved in understanding the function of individual genes associated with sugar metabolism and sugar transport in non-fruit crops, there is less known about the sugar transporters and metabolic enzymes responsible for sugar accumulation in fruit crop species. This review identifies knowledge gaps and can serve as a foundation for future studies, with comprehensive updates focusing on (1) the physiological roles of the metabolic enzymes and sugar transporters responsible for sugar allocation and partitioning and that contribute to sugar accumulation in fruit crops; and (2) the molecular mechanisms underlying the transcriptional and posttranslational regulation of sugar transport and metabolism. We also provide insights into the challenges and future directions of studies on sugar transporters and metabolic enzymes and name several promising genes that should be targeted with gene editing in the pursuit of optimized sugar allocation and partitioning to enhance sugar accumulation in fruits.

ClSnRK2.3 negatively regulates watermelon fruit ripening and sugar accumulation.

Watermelon (Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid (ABA) signaling pathway gene ClSnRK2.3 might influence watermelon fruit ripening. However, the molecular mechanisms are unclear. Here, we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator in fruit ripening. Overexpression (OE) of ClSnRK2.3 significantly delayed watermelon fruit ripening and suppressed the accumulation of sucrose, ABA and gibberellin GA4 . Furthermore, we determined that the pyrophosphate-dependent phosphofructokinase (ClPFP1) in sugar metabolism pathway and GA biosynthesis enzyme GA20 oxidase (ClGA20ox) could be phosphorylated by ClSnRK2.3 and thereby resulting in accelerated protein degradation in OE lines and finally led to low levels of sucrose and GA4 . Besides that, ClSnRK2.3 phosphorylated homeodomain-leucine zipper protein (ClHAT1) and protected it from degradation to suppress the expression of the ABA biosynthesis gene 9'-cis-epoxycarotenoid dioxygenase 3 (ClNCED3). These results indicated that ClSnRK2.3 negatively regulated watermelon fruit ripening by manipulating the biosynthesis of sucrose, ABA and GA4 . Altogether, these findings revealed a novel regulatory mechanism in non-climacteric fruit development and ripening.

Exploring the Developmental Progression of Endosperm Cavity Formation in Maize Grain and the Underlying Molecular Basis Using X-Ray Tomography and Genome Wide Association Study.

Endosperm cavity (EC) in maize grain reduces yield and causes grain breakage during mechanical harvesting, hence representing a major problem in the maize industry. Despite this, little is known regarding the biological processes governing EC formation. Here, we attempted to address this issue by (i) determining the spatial and temporal progression of EC in a non-invasive manner and (ii) identifying candidate genes that may be linked to the formation of EC by using a genome wide association study (GWAS). Visualization and measurement using X-ray micro-computed tomography established that EC first appeared at the central starch endosperm at about 12 days after pollination (DAP) and became enlarged thereafter. GWAS-based screening of a panel of 299 inbred lines with a wide range of EC size identified nine candidate genes that showed significant association with EC formation. Most of the candidate genes exhibited a decrease at 12 DAP, coinciding with the timing of EC appearance. Among them, ZmMrp11 was annotated as a member encoding a multidrug resistance-associated protein that has been shown in other studies to sequestrate toxic metabolites from the cytosol to the vacuole, thereby detoxifying the cellular environment. This, together with the reduced expression of ZmMrp11 in maize grains from 12 DAP, prompted us to propose that the low expression of ZmMrp11 may block cellular detoxification in the maize endosperm cells, leading to cell death and ultimately the formation of EC.

A transcriptional landscape underlying sugar import for grain set in maize.

Developing seed depends on sugar supply for its growth and yield formation. Maize (Zea mays L.) produces the largest grains among cereals. However, there is a lack of holistic understanding of the transcriptional landscape of genes controlling sucrose transport to, and utilization within, maize grains. By performing in-depth data mining of spatio-temporal transcriptomes coupled with histological and heterologous functional analyses, we identified transporter genes specifically expressed in the maternal-filial interface, including (i) ZmSWEET11/13b in the placento-chalazal zone, where sucrose is exported into the apoplasmic space, and (ii) ZmSTP3, ZmSWEET3a/4c (monosaccharide transporters), ZmSUT1, and ZmSWEET11/13a (sucrose transporters) in the basal endosperm transfer cells for retrieval of apoplasmic sucrose or hexoses after hydrolysis by extracellular invertase. In the embryo and its surrounding regions, an embryo-localized ZmSUT4 and a cohort of ZmSWEETs were specifically expressed. Interestingly, drought repressed those ZmSWEETs likely exporting sucrose but enhanced the expression of most transporter genes for uptake of apoplasmic sugars. Importantly, this drought-induced fluctuation in gene expression was largely attenuated by an increased C supply via controlled pollination, indicating that the altered gene expression is conditioned by C availability. Based on the analyses above, we proposed a holistic model on the spatio-temporal expression of genes that likely govern sugar transport and utilization across maize maternal and endosperm and embryo tissues during the critical stage of grain set. Collectively, the findings represent an advancement towards a holistic understanding of the transcriptional landscape underlying post-phloem sugar transport in maize grain and indicate that the drought-induced changes in gene expression are attributable to low C status.

Identification of transcription factors controlling cell wall invertase gene expression for reproductive development via bioinformatic and transgenic analyses.

Cell wall invertase (CWIN) hydrolyses sucrose into glucose and fructose in the extracellular matrix and plays crucial roles in assimilate partitioning and sugar signalling. However, the molecular regulators controlling CWIN gene transcription remain unknown. As the first step to address this issue, we performed bioinformatic and transgenic studies, which identified a cohort of transcription factors (TFs) modulating CWIN gene expression in Arabidopsis thaliana. Comprehensive bioinformatic analyses identified 18 TFs as putative regulators of the expression of AtCWIN2 and AtCWIN4 that are predominantly expressed in Arabidopsis reproductive organs. Among them, MYB21, ARF6, ARF8, AP3 and CRC were subsequently shown to be the most likely regulators of CWIN gene expression based on molecular characterization of the respective mutant of each candidate TF. More specifically, the obtained data indicate that ARF6, ARF8 and MYB21 regulate CWIN2 expression in the anthers and CWIN4 in nectaries, anthers and petals, whereas AP3 and CRC were determined primarily to regulate the transcriptional activity of CWIN4. TF-promoter interaction assays demonstrated that ARF6 and ARF8 directly control CWIN2 and CWIN4 transcription with AP3 activating CWIN4. The involvement of ARF8 in regulating CWIN4 expression was further supported by the finding that enhanced CWIN4 expression partially recovered the short silique phenotype displayed by the arf8-3 mutant. The identification of the five TFs regulating CWIN expression serves as a launching pad for future studies to dissect the upstream molecular network underpinning the transcription of CWINs and provides a new avenue, potentially, to engineer assimilate allocation and reproductive development for improving seed yield.

Dissecting the phenotypic components and genetic architecture of maize stem vascular bundles using high-throughput phenotypic analysis.

High-throughput phenotyping is increasingly becoming an important tool for rapid advancement of genetic gain in breeding programmes. Manual phenotyping of vascular bundles is tedious and time-consuming, which lags behind the rapid development of functional genomics in maize. More robust and automated techniques of phenotyping vascular bundles traits at high-throughput are urgently needed for large crop populations. In this study, we developed a standard process for stem micro-CT data acquisition and an automatic CT image process pipeline to obtain vascular bundle traits of stems including geometry-related, morphology-related and distribution-related traits. Next, we analysed the phenotypic variation of stem vascular bundles between natural population subgroup (480 inbred lines) based on 48 comprehensively phenotypic information. Also, the first database for stem micro-phenotypes, MaizeSPD, was established, storing 554 pieces of basic information of maize inbred lines, 523 pieces of experimental information, 1008 pieces of CT scanning images and processed images, and 24 192 pieces of phenotypic data. Combined with genome-wide association studies (GWASs), a total of 1562 significant single nucleotide polymorphism (SNPs) were identified for 30 stem micro-phenotypic traits, and 84 unique genes of 20 traits such as VBNum, VBAvArea and PZVBDensity were detected. Candidate genes identified by GWAS mainly encode enzymes involved in cell wall metabolism, transcription factors, protein kinase and protein related to plant signal transduction and stress response. The results presented here will advance our knowledge about phenotypic trait components of stem vascular bundles and provide useful information for understanding the genetic controls of vascular bundle formation and development.

Cell Wall Invertase Is Essential for Ovule Development through Sugar Signaling Rather Than Provision of Carbon Nutrients.

Ovule formation is essential for realizing crop yield because it determines seed number. The underlying molecular mechanism, however, remains elusive. Here, we show that cell wall invertase (CWIN) functions as a positive regulator of ovule initiation in Arabidopsis (Arabidopsis thaliana). In situ hybridization revealed that CWIN2 and CWIN4 were expressed at the placenta region where ovule primordia initiated. Specific silencing of CWIN2 and CWIN4 using targeted artificial microRNA driven by an ovule-specific SEEDSTICK promoter (pSTK) resulted in a substantial reduction of CWIN transcript and activity, which blocked ovule initiation and aggravated ovule abortion. There was no induction of carbon (C) starvation genes in the transgenic lines, and supplementing newly forming floral buds with extra C failed to recover the ovule phenotype. This indicates that suppression of CWIN did not lead to C starvation. A group of hexose transporters was downregulated in the transgenic plants. Among them, two representative ones were spatially coexpressed with CWIN2 and CWIN4, suggesting a coupling between CWIN and hexose transporters for ovule initiation. RNA-sequencing analysis identified differentially expressed genes encoding putative extracellular receptor-like kinases, MADS-box transcription factors, including STK, and early auxin response genes in response to CWIN-silencing. Our data demonstrate the essential role of CWIN in ovule initiation, which is most likely to occur through sugar signaling instead of C nutrient contribution. We propose that CWIN-mediated sugar signaling may be perceived by, and transmitted through, hexose transporters or receptor-like kinases to regulate ovule formation by modulating downstream auxin signaling and MADS-box transcription factors.

Cell Wall Invertase and Sugar Transporters Are Differentially Activated in Tomato Styles and Ovaries During Pollination and Fertilization.

Flowering plants depend on pollination and fertilization to activate the transition from ovule to seed and ovary to fruit, namely seed and fruit set, which are key for completing the plant life cycle and realizing crop yield potential. These processes are highly energy consuming and rely on the efficient use of sucrose as the major nutrient and energy source. However, it remains elusive as how sucrose imported into and utilizated within the female reproductive organ is regulated in response to pollination and fertilization. Here, we explored this issue in tomato by focusing on genes encoding cell wall invertase (CWIN) and sugar transporters, which are major players in sucrose phloem unloading, and sink development. The transcript level of a major CWIN gene, LIN5, and CWIN activity were significantly increased in style at 4 h after pollination (HAP) in comparison with that in the non-pollination control, and this was sustained at 2 days after pollination (DAP). In the ovaries, however, CWIN activity and LIN5 expression did not increase until 2 DAP when fertilization occurred. Interestingly, a CWIN inhibitor gene INVINH1 was repressed in the pollinated style at 2 DAP. In response to pollination, the style exhibited increased expressions of genes encoding hexose transporters, SlHT1, 2, SlSWEET5b, and sucrose transporters SlSUT1, 2, and 4 from 4 HAP to 2 DAP. Upon fertilization, SlSUT1 and SlHT1 and 2, but not SlSWEETs, were also stimulated in fruitlets at 2 DAP. Together, the findings reveal that styles respond promptly and more broadly to pollination for activation of CWIN and sugar transporters to fuel pollen tube elongation, whereas the ovaries do not exhibit activation for some of these genes until fertilization occurs. HIGHLIGHTS: Expression of genes encoding cell wall invertases and sugar transporters was stimulated in pollinated style and fertilized ovaries in tomato.

Cell wall invertase as a regulator in determining sequential development of endosperm and embryo through glucose signaling early in seed development.

Seed development depends on coordination among embryo, endosperm and seed coat. Endosperm undergoes nuclear division soon after fertilization, whereas embryo remains quiescent for a while. Such a developmental sequence is of great importance for proper seed development. However, the underlying mechanism remains unclear. Recent results on the cellular domain- and stage-specific expression of invertase genes in cotton and Arabidopsis revealed that cell wall invertase may positively and specifically regulate nuclear division of endosperm after fertilization, thereby playing a role in determining the sequential development of endosperm and embryo, probably through glucose signaling.