The Role of Strigolactones in the Regulation of Root System Architecture in Grapevine (Vitis vinifera L.) in Response to Root-Restriction Cultivation.

This study investigated the effects of root-restriction cultivation on the root architecture, endogenous strigolactone (SL) content, and SL-related genes expression in grapevine (Vitis vinifera L.). In addition, we clarified the effects of synthetic SL analog GR24 application on grapevine roots to explore the role of SLs in their development. The results showed that the root architecture changed significantly under root-restriction cultivation. At 40 days after transplantation (DAT), the contents of two types of SLs in roots under root restriction were both significantly lower than that in roots of the control. SL content was significantly positively correlated with the expression levels of VvCCD8 and VvD27, indicating that they play vital roles in SLs synthesis. After GR24 treatment for 20 days, the root length was significantly shorter than in the control. A low concentration (0.1 µM) of GR24 significantly reduced the root diameter and increased the fine-root density, while a high concentration (10 µM) of GR24 significantly reduced the lateral root (LR) length and increased the LR density. Concomitantly, GR24 (0.1 µM) reduced endogenous SL content. After GR24 treatment for 5 days, the total content of two tested SLs was highly positively correlated with the expression levels of VvDAD2, whereas it was highly negatively correlated with VvSMAXL4 at 20 days after GR24 treatment. This study helps to clarify the internal mechanism of root-restriction cultivation affecting the changes in grapevine root architecture, as well as further explore the important role of SLs in the growth of grapevine roots in response to root-restriction treatment.

Small RNA Sequencing Analysis of miRNA Expression Reveals Novel Insihts into Root Formation under Root Restriction Cultivation in Grapevine (Vitis vinifera L.).

Root restriction cultivation (RRC) can influence plant root architecture, but its root phenotypic changes and molecular mechanisms are still unknown. In this study, phenotype observations of grapevine root under RRC and control cultivation (nRC) at 12 time points were conducted, and the root phenotype showed an increase of adventitious and lateral root numbers and root tip degeneration after RRC cultivation from 70 days after planting (DAP). The 70 and 125 DAP sampling of two different cultivations, named nR70, RR70, nR125, and RR125, were selected for small RNA sequencing. A total of 153 known miRNAs and 119 predicted novel miRNAs were obtained. Furthermore, BLAST was used to predict the novel miRNAs with miRBase databases using the default parameters; 96 of the 119 predicted novel miRNAs were similar to other species, and the remaining 23 grapevine-specific novel miRNAs were obtained. There were 26, 33, 26, and 32 miRNAs that were differentially expressed in different comparison groups (RR70 vs. nR70, RR125 vs. nR125, nR125 vs. nR70 and RR125 vs. RR70). Target genes prediction of differentially expressed miRNAs was annotated on a variety of biological processes, and 24 participated in root development. Moreover, multiple miRNAs were found to jointly regulate lateral root development under root restriction conditions. The miRNA expression pattern comparison between RRC and nRC may provide a framework for the future analysis of miRNAs associated with root development in grapevine.

Comparative Metabolic Profiling of Grape Skin Tissue along Grapevine Berry Developmental Stages Reveals Systematic Influences of Root Restriction on Skin Metabolome.

This research aimed to comparatively evaluate the influences of root restriction (RR) cultivation and traditional cultivation (RC) on grape berry skin metabolomics using a non-targeted metabolomics method. Two-hundred-and-ninety-one metabolites were annotated and the kinetics analyses showed that berry skin metabolome is stage- and cultivation-dependent. Our results showed that RR influences significantly the metabolomes of berry skin tissues, particularly on secondary metabolism, and that this effect is more obvious at pre-veraison stage, which was evidenced by the early and fast metabolic shift from primary to secondary metabolism. Altogether, this study provided an insight into metabolic adaptation of berry skin to RR stress and expanded general understanding of berry development.

Transcriptomic Analyses of Root Restriction Effects on Phytohormone Content and Signal Transduction during Grape Berry Development and Ripening.

Phytohormones strongly influence growth, development and nutritional quality of agricultural products by modulating molecular and biochemical changes. The purpose of the present study was to investigate the influence of root restriction (RR) treatment on the dynamic changes of main phytohormones during the berry development and ripening of "Summer Black" early ripening seedless grape (Vitis vinifera × V. labrusca), and to analyze the changes in the biosynthesis and signal transduction pathways of phytohormones by transcriptomics. Enzyme-linked immunosorbent assay (ELISA) and Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS) were used to quantify the phytohormone levels, and RNA-Seq was used to analyze the transcript abundance. The results showed that 23 transcripts involved in the phytohormone biosynthesis and 34 transcripts involved in the signal transduction pathways were significantly changed by RR treatment. RR also increased abscisic acid, brassinosteroid, ethylene, jasmonic acid and salicylic acid levels, while decreasing auxin, cytokinin, and gibberellin contents. The results of the present study suggest that RR treatment can accelerate the grape ripening process, and specific candidate genes were identified for further functional analysis.

Comparative Transcriptomic Analysis of Grape Berry in Response to Root Restriction during Developmental Stages.

Root restriction improved berry quality by being involved in diverse aspects of grapevine life. However, the molecular mechanism driving this process is not understood very well. In this study, the 'Summer Black' grape berry (Vitis vinifera × V. labrusca) under root restriction was investigated, which showed an increase of total soluble solids (TSS), color index of red grapes (CIRG) value, anthocyanins accumulation, total phenolics and total procyanidins contents during berry development compared with those in control berries. The transcriptomic changes induced by root restriction in 'Summer Black' grape over the course of berry development were analyzed by RNA-Seq method. A total of 29,971 genes were generated in 'Summer Black' grape berry during development, among which, 1606 genes were significantly responded to root restriction. Furthermore, 1264, 313, 141, 246 and 19 sequences were significantly changed at S1, S2, S3, S4 and S5 sample points, respectively. The gene (VIT_04s0023g02290) predicted as a salicylate O-methyltransferase was differentially expressed in all developmental stages. Gene Ontology (GO) enrichment showed that response to organic nitrogen, response to endogenous stimulus, flavonoid metabolic process, phenylpropanoid biosynthetic process and cell wall macromolecule metabolic process were the main significant differential categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed plant-pathogen interaction, plant hormone signal transduction, flavone and flavonol biosynthesis, flavonoid biosynthesis and glucosinolate biosynthesis were the main significant differential pathways. The results of the present study provided a genetic base for the understanding of grape berry fruit quality improvement under root restriction.

Sugar accumulation may be regulated by a transcriptional cascade of ABA-VvGRIP55-VvMYB15-VvSWEET15 in grape berries under root restriction.

In the southern of China, precipitation is abundant during the grape growing season, which results in lower sugar content, and finally reduces the quality and yield of grape berries and leads to lower economic benefits. The root restriction cultivation method is an important abiotic stress that limits the disordered growth and development of roots, and it favors the accumulation of sugar and abscisic acid. However, the relationship between ABA and sugar accumulation under root restriction remains unclear. Here, we tested the expression levels of several transcription factors and sugar metabolism-related genes and found that root restriction cultivation could induce higher expression of VvMYB15 and VvSWEET15. The VvMYB15 transcription factor was found to bind to the promoter of VvSWEET15 and activate its expression, furthermore, transient overexpression of VvMYB15 in strawberry fruits and grape berries can promote sugar accumulation and increase the expression level of sugar metabolism-related genes, indicating that VvMYB15 is a positive regulator of sugar accumulation. In addition, the endogenous ABA content and expression level of VvGRIP55, which is highly responsive to ABA, were significantly increased under root restriction, and VvGRIP55 could bind to the promoter of VvMYB15 and activate its expression. Therefore, our results demonstrated that the ABA-responsive factor VvGRIP55 can promote sugar accumulation through VvMYB15 and VvSWEET15, suggesting a mechanism by which ABA regulates sugar accumulation under root restriction.

Transcriptomic Analysis of Root Restriction Effects on the Primary Metabolites during Grape Berry Development and Ripening.

Root restriction (RR) has been reported to enhance grape berry quality in diverse aspects of grape life. In this study, RR-induced increases in the main primary metabolites in the grape berry and the expression of their related genes were studied at different developmental stages. Mainly the transcriptomic and metabolomic level were analyzed using 'Summer Black' grape berry as a material. The main results were as follows: A total of 11 transcripts involved in the primary metabolic pathways were significantly changed by the RR treatment. Metabolites such as sugars, organic acids, amino acids, starch, pectin, and cellulose were qualitatively and quantitatively analyzed along with their metabolic pathways. Sucrose synthase (VIT_07s0005g00750, VIT_11s0016g00470) and sucrose phosphate synthase (VIT_18s0089g00410) were inferred to play critical roles in the accumulation of starch, sucrose, glucose, and fructose, which was induced by the RR treatment. RR treatment also promoted the malic acid and tartaric acid accumulation in the young berry. In addition, the grape berries after the RR treatment tended to have lower pectin and cellulose content.

Corrigendum: VvMYB15 and VvWRKY40 Positively Co-regulated Anthocyanin Biosynthesis in Grape Berries in Response to Root Restriction.

[This corrects the article DOI: 10.3389/fpls.2021.789002.].

Integrative Investigation of Root-Related mRNAs, lncRNAs and circRNAs of "Muscat Hamburg" (Vitis vinifera L.) Grapevine in Response to Root Restriction through Transcriptomic Analyses.

Root restriction is a physical and ecological cultivation mode which restricts plant roots into a limited container to regulate vegetative and reproduction growth by reshaping root architecture. However, little is known about related molecular mechanisms. To uncover the root-related regulatory network of endogenous RNAs under root restriction cultivation (referred to RR), transcriptome-wide analyses of mRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) involved in root development were performed. During root development, RR treatment had a positive effect on root weight, typically, young roots were significantly higher than conventional cultivation (referred to NR) treatment, suggesting that root architecture reconstruction under RR was attributed to the vigorous induction into lateral roots. Furthermore, a total of 26,588 mRNAs, 1971 lncRNAs, and 2615 circRNAs were identified in root of annual "Muscat Hamburg" grapevine by the transcriptomic analyses. The expression profile of mRNAs, lncRNAs and circRNA were further confirmed by the quantitative real-time PCR (RT-qPCR). Gene ontology enrichment analysis showed that a majority of the differentially expressed mRNAs, lncRNAs and circRNAs were enriched into the categories of cellular process, metabolic process, cell part, binding, and catalytic activity. In addition, the regulatory network of endogenous RNAs was then constructed by the prediction of lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA network, implying that these RNAs play significant regulatory roles for root architecture shaping in response to root restriction. Our results, for the first time, the regulatory network of competitive endogenous RNAs (ceRNAs) functions of lncRNA and circRNA was integrated, and a basis for studying the potential functions of non-coding RNAs (ncRNAs) during root development of grapevine was provided.

Regulatory effect of root restriction on aroma quality of Red Alexandria grape.

To systematically study the impact of root restriction (RR) on the aroma quality of grape berry, in this study, free and bound compounds were investigated in 'Red Alexandria' grape skin and pulp produced with and without RR during development and ripening. Compared with the control, RR advanced the initiation of free-terpene synthesis and increased their concentrations at 14-18 weeks post-flowering (wpf) by promoting the conversion of bound terpenes to free terpenes. In addition, RR significantly regulated the aromatic series at 14-18 wpf and advanced the date of aroma maturation. Network analyses indicated that the correlations among bound compounds were more conserved than those among free compounds, and the skin network displayed tight coordination compared with the pulp network. Terpenes were highly intercorrelated and played a core role in these networks. Finally, 10 bound compounds in pulp were screened out as indicators of the developmental timing of grape.

Transcriptome analysis revealed the expression levels of genes related to abscisic acid and auxin biosynthesis in grapevine (Vitis vinifera L.) under root restriction.

The root system is essential for the stable growth of plants. Roots help anchor plants in the soil and play a crucial role in water uptake, mineral nutrient absorption and endogenous phytohormone formation. Root-restriction (RR) cultivation, a powerful technique, confines plant roots to a specific soil space. In the present study, roots of one-year-old "Muscat Hamburg" grapevine under RR and control (nR) treatments harvested at 70 and 125 days after planting were used for transcriptome sequencing, and in total, 2031 (nR7 vs. nR12), 1445 (RR7 vs. RR12), 1532 (nR7 vs. RR7), and 2799 (nR12 vs. RR12) differentially expressed genes (DEGs) were identified. Gene Ontology (GO) enrichment analysis demonstrated that there were several genes involved in the response to different phytohormones, including abscisic acid (ABA), auxin (IAA), ethylene (ETH), gibberellins (GAs), and cytokinins (CTKs). Among them, multiple genes, such as PIN2 and ERF113, are involved in regulating vital plant movements by various phytohormone pathways. Moreover, following RR cultivation, DEGs were enriched in the biological processes of plant-type secondary cell wall biosynthesis, the defense response, programmed cell death involved in cell development, and the oxalate metabolic process. Furthermore, through a combined analysis of the transcriptome and previously published microRNA (miRNA) sequencing results, we found that multiple differentially expressed miRNAs (DEMs) and DEG combinations in different comparison groups exhibited opposite trends, indicating that the expression levels of miRNAs and their target genes were negatively correlated. Furthermore, RR treatment indeed significantly increased the ABA content at 125 days after planting and significantly decreased the IAA content at 70 days after planting. Under RR cultivation, most ABA biosynthesis-related genes were upregulated, while most IAA biosynthesis-related genes were downregulated. These findings lay a solid foundation for further establishing the network through which miRNAs regulate grapevine root development through target genes and for further exploring the molecular mechanism through which endogenous ABA and IAA regulate root architecture development in grapevine.

VvMYB15 and VvWRKY40 Positively Co-regulated Anthocyanin Biosynthesis in Grape Berries in Response to Root Restriction.

In most grapevine planting regions, especially in south of China, plenty of rainfall and high water level underground are the characteristic of the area, a series of problem during fruit ripening easily caused poor color quality. Thereby affecting fruit quality, yield and economic benefits. The accumulation of anthocyanin is regulated by transcriptional regulatory factor and a series of cultivation measures, root restriction can make plants in the environment of stress and stress relief, root restriction induced the higher expression of VvMYB15 and VvWRKY40, and consistent with anthocyanin accumulation. Whether and how root restriction-inducible VvMYB15 and VvWRKY40 transcription factor regulate anthocyanin synthesis in grape berry is still unclear. In this study, we identified that the transient overexpression of VvMYB15 and VvWRKY40 alone or both in strawberry fruits and grape berries can promote anthocyanin accumulation and increase the expression level of anthocyanin biosynthetic genes, indicating VvMYB15 and VvWRKY40 play a positive regulator of anthocyanin biosynthesis. Furthermore, we confirmed that both VvMYB15 and VvWRKY40 specifically bind to the promoter region of VvF3'5'H and VvUFGT, and the expression of VvF3'5'H and VvUFGT is further activated through the heterodimer formation between VvMYB15 and VvWRKY40. Finally, we confirmed that VvMYB15 promoted anthocyanin accumulation by interacting with VvWRKY40 in grape berries, our findings provide insights into a mechanism involving the synergistic regulation of root restriction-dependent coloration and biosynthesis via a VvMYB15 and VvWRKY40 alone or both in grape berries.

Comparative Metabolic Profiling of Grape Pulp during the Growth Process Reveals Systematic Influences under Root Restriction.

The compositions and contents of metabolites in the pulp tissue play critical roles in the fruit quality for table grape. In this study, the effects of root restriction (RR) on the primary and secondary metabolites of pulp tissue at five developmental stages were studied at the metabolomics level, using "Red Alexandria" grape berry (Vitis vinifera L.) as materials. The main results were as follows: 283 metabolites were annotated by using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS); 28 and 16 primary metabolites contents were increased and decreased, and 11 and 19 secondary metabolites contents were increased and decreased, respectively, along the berry development; RR significantly decreased 12 metabolites (four amino acids and derivatives, three organic acids, four flavonoids and one other compound) contents, and improved 40 metabolites (22 amino acids and derivatives, six nucleotides, four carbohydrates, four cofactors, three cinnamic acids and one other compound) accumulation at the different developmental stages. Altogether, our study would be helpful to increase our understanding of grape berry's responses to RR stress.

Transcriptomic Analysis of Root Restriction Effects on Phenolic Metabolites during Grape Berry Development and Ripening.

In the present study, the effects of root restriction (RR) on the main phenolic metabolites and the related gene expression at different developmental stages were studied at the transcriptomic and metabolomic levels in "Summer Black" grape berries (Vitis vinifera × Vitis labrusca). The results were as follows: seven phenolic acid compounds, three stilbene compounds, nine flavonol compounds, 10 anthocyanin compounds, and 24 proanthocyanidin compounds were identified by ultra-performance liquid chromatography-high-resolution mass spectrometry. RR treatment significantly promoted the biosynthesis of phenolic acid, trans-resveratrol, flavonol, and anthocyanin and also affected the proanthocyanidin content, which was elevated in the early developmental stages and then reduced in the late developmental stages. The functional genes for phenylalanine ammonia-lyase, trans-cinnamate 4-monooxygenase, 4-coumarate-CoA ligase, shikimate O-hydroxycinnamoyl transferase, chalcone synthase, chalcone isomerase, stilbene synthase, flavonoid 3',5'-hydroxylase, anthocyanidin 3-O-glucosyltransferase, and the transcription factors MYBA1, MYBA2, MYBA3, and MYBA22 were inferred to play critical roles in the changes regulated by RR treatment.

Transcriptomic Analyses of Ascorbic Acid and Carotenoid Metabolites Influenced by Root Restriction during Grape Berry Development and Ripening.

Ascorbic acid (AsA) and carotenoids are recognized as crucial metabolites for various biological processes in plants. The contents of AsA and carotenoids in fruits are influenced by external environmental stimuli, such as water, temperature, light, and hormones. However, it is still not clear whether it can be affected by root restriction (RR) treatment. In this study, "Summer Black" grape berries (Vitis vinifera × V. labrusca) under RR and control treatments during development and ripening were used as materials. The results showed that RR significantly increased the contents of AsA, and the transcript VIT_08s0040g03150 related to AsA recycling pathways may be the main regulator for AsA. Similarly, the contents of most of the carotenoids at the earlier stages significantly increased by RR; the enzyme crtB encoded by VIT_12s0028g00960 and the enzyme crtZ encoded by VIT_02s0025g00240 and VIT_16s0050g01090 were inferred to play major roles in the carotenoid metabolic pathways.

Elevated CO2 alters deployment of roots in "small" growth containers.

Previously we examined how limited rooting space and nutrient supply influenced plant growth under elevated atmospheric CO2 concentrations (McConnaughay et al. 1993). We demonstrated that plant growth enhancement under elevated CO2 was influenced more by the concentration of nutrients added to growth containers than to either the total nutrient content per pot or amount or the dimensions of available rooting space. To gain insight into how elevated CO2 atmospheres affect how plants utilize available belowground space when rooting space and nutrient supply are limited we measured the deployment of roots within pots through time. Contrary to aboveground responses, patterns of belowground deployment were most strongly influenced by elevated CO2 in pots of different volume and shape. Further, elevated CO2 conditions interacted differently with limited belowground space for the two species we studied,Abutilon theophrasti, a C3 dicot with a deep taproot, andSetaria faberii, a C4 monocot with a shallow fibrous root system. ForSetaria, elevated CO2 increased the size of the largest region of low root density at the pot surface in larger rooting volumes independent of nutrient content, thereby decreasing their efficiency of deployment. ForAbutilon, plants responded to elevated CO2 concentrations by equalizing the pattern of deployment in all the pots. Nutrient concentration, and not pot size or shape, greatly influenced the density of root growth. Root densities forAbutilon andSetaria were similar to those observed in field conditions, for annual dicots and monocots respectively, suggesting that studies using pots may successfully mimic natural conditions.

Limitations to CO2-induced growth enhancement in pot studies.

Recently, it has been suggested that small pots may reduce or eliminate plant responses to enriched CO2 atmospheres due to root restriction. While smaller pot volumes provide less physical space available for root growth, they also provide less nutrients. Reduced nutrient availability alone may reduce growth enhancement under elevated CO2. To investigate the relative importance of limited physical rooting space separate from and in conjunction with soil nutrients, we grew plants at ambient and double-ambient CO2 levels in growth containers of varied volume, shape, nutrient concentration, and total nutrient content. Two species (Abutilon theophrasti, a C3 dicot with a deep tap root andSetaria faberii, a C4 monocot with a shallow diffuse root system) were selected for their contrasting physiology and root architecture. Shoot demography was determined weekly and biomass was determined after eight and ten weeks of growth. Increasing total nutrients, either by increasing nutrient concentration or by increasing pot size, increased plant growth. Further, increasing pot size while maintaining equal total nutrients per pot resulted in increased total biomass for both species. CO2-induced growth and reproductive yield enhancements were greatest in pots with high nutrient concentrations, regardless of total nutrient content or pot size, and were also mediated by the shape of the pot. CO2-induced growth and reproductive yield enhancements were unaffected by pot size (growth) or were greater in small pots (reproductive yield), regardless of total nutrient content, contrary to predictions based on earlier studies. These results suggest that several aspects of growth conditions within pots may influence the CO2 responses of plants; pot size, pot shape, the concentration and total amount of nutrient additions to pots may lead to over-or underestimates of the CO2 responses of real-world plants.