Challenges and prospects of new plant breeding techniques for GABA improvement in crops: tomato as an example

[EN] Over the last seven decades, gamma-aminobutyric acid (GABA) has attracted great attention from scientists for its ubiquity in plants, animals and microorganisms and for its physiological implications as a signaling molecule involved in multiple pathways and processes. Recently, the food and pharmaceutical industries have also shown significantly increased interest in GABA, because of its great potential benefits for human health and the consumer demand for health-promoting functional compounds, resulting in the release of a plethora of GABA-enriched products. Nevertheless, many crop species accumulate appreciable GABA levels in their edible parts and could help to meet the daily recommended intake of GABA for promoting positive health effects. Therefore, plant breeders are devoting much effort into breeding elite varieties with improved GABA contents. In this regard, tomato (Solanum lycopersicum), the most produced and consumed vegetable worldwide and a fruit-bearing model crop, has received much consideration for its accumulation of remarkable GABA levels. Although many different strategies have been implemented, from classical crossbreeding to induced mutagenesis, new plant breeding techniques (NPBTs) have achieved the best GABA accumulation results in red ripe tomato fruits along with shedding light on GABA metabolism and gene functions. In this review, we summarize, analyze and compare all the studies that have substantially contributed to tomato GABA breeding with further discussion and proposals regarding the most recent NPBTs that could bring this process to the next level of precision and efficiency. This document also provides guidelines with which researchers of other crops might take advantage of the progress achieved in tomato for more efficient GABA breeding programsPG is grateful to the Japanese Society for the Promotion of Science for the JSPS postdoctoral grant FY2019-P19105Gramazio, P.; Takayama, M.; Ezura, H. (2020). Challenges and prospects of new plant breeding techniques for GABA improvement in crops: tomato as an example. Frontiers in Plant Science. 11:1-16. https://doi.org/10.3389/fpls.2020.577980S1161

Targeted modification of CmACO1 by CRISPR/Cas9 extends the shelf-life of Cucumis melo var. reticulatus melon

The gaseous plant hormone ethylene is a regulator of fruit shelf-life, one of the essential traits in fruits. Extending fruit shelf-life reduces food loss, thereby expected to contribute to food security. The enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) is the final step of the ethylene production pathway. Its suppression via antisense technology has been demonstrated to extend the shelf-life of melon, apple, and papaya. Genome editing technology is an innovative technique for plant breeding. Because the genome editing technology would not leave the exogenous genes in the final crop products, the crops via genome editing can be considered non-genetically modified yields; compared to conventional breeding, such as mutation breeding, the breeding term would be expected to be relatively short. These points include the advantage of this technique in utilization for commercial applications. We attempted to extend the shelf-life of the Japanese luxury melon (Cucumis melo var. reticulatus, ‘Harukei-3’) via modification of the ethylene synthesis pathway with the genome editing technology, CRISPR/Cas9 system. The Melonet-DB (https://melonet-db.dna.affrc.go.jp/ap/top) showed that the melon genome had the five CmACOs and the gene CmACO1 predominantly expressed in harvested fruits. From this information, CmACO1 was expected to be a key gene for shelf-life in melons. Based on this information, the CmACO1 was selected as the target of the CRISPR/Cas9 system and introduced the mutation. The final product of this melon did not have any exogenous genes. The mutation was inherited for at least two generations. In the T2 generation, the fruit phenotypes 14 days after harvest were as follows: ethylene production was reduced to one-tenth that of the wild type, pericarp colour remained green, and higher fruit firmness. Early fermentation of the fresh fruit was observed in the wild-type fruit but not in the mutant. These results show that CmACO1 knockout via CRISPR/Cas9 extended the melon’s shelf-life. Moreover, our results suggest that genome editing technology would reduce food loss and contribute to food security

Improved G-AgarTrap: A highly efficient transformation method for intact gemmalings of the liverwort Marchantia polymorpha

Liverworts are key species for studies of plant evolution, occupying a basal position among the land plants. Marchantia polymorpha has emerged as a highly studied model liverwort, and many relevant techniques, including genetic transformation, have been established for this species. Agrobacterium-mediated transformation is widely used in many plant species because of its low cost. Recently, we developed a simplified Agrobacterium-mediated method for transforming M. polymorpha, known as AgarTrap (agar-utilized transformation with pouring solutions). The AgarTrap procedure, which involves culturing the liverwort tissue in various solutions on a single solid medium, yields up to a hundred independent transformants. AgarTrap is a simple procedure, requiring minimal expertise, cost, and time. Here, we investigated four factors that influence AgarTrap transformation efficiency: (1) humidity, (2) surfactant in the transformation buffer, (3) Agrobacterium strain, and (4) light/dark condition. We adapted the AgarTrap protocol for transforming intact gemmalings, achieving an exceptionally high transformation efficiency of 97%. The improved AgarTrap method will enhance the molecular biological study of M. polymorpha. Furthermore, this method provides new possibilities for improving transformation techniques for a variety of plant species

SEXUAL STERILITY is Essential for Both Male and Female Gametogenesis in Tomato

Gametogenesis is a key step in the production of ovules or pollen in higher plants. The molecular aspects of gametogenesis are well characterized in the model plant Arabidopsis; however, little information is known in tomato, which is a model plant for fleshy fruit development. In this study, we characterized a tomato (Solanum lycopersicum L.) γ-ray mutant, sexual sterility (Slses), that exhibited both male and female sterility. Morphological analysis revealed that the Slses mutant forms incomplete ovules and wilted anthers devoid of pollen grains at the anthesis stage. Genetic and next-generation sequencing analyses revealed that the Slses mutant carried a 13 bp deletion within the first exon of a homolog of SPOROCYTELESS/NOZZLE (SPL/NZZ), which plays an important role in gametogenesis in Arabidopsis. Complementation analysis in which the complete SlSES genomic region was introduced into the Slses mutant fully restored normal phenotypes, demonstrating that Solyc07g063670 is responsible for the Slses mutation. SlSES probably act as a transcriptional repressor because of an EAR motif at the C-terminal region. Gene expression levels of WUSCHEL (SlWUS) and INNER NO OUTER (SlINO), both of which are required for ovule development, were dramatically reduced in the early stages of pistil development in the Slses mutant, suggesting a positive regulatory role for SlSES in the transcription of gametogenesis genes and differences in the regulation of INO (SlINO) and integument development by SPL/NZZ (SLSES) between Arabidopsis and tomato. Taken together, our results indicate that SlSES is a novel tomato gametogenesis gene essential for both male and female gametogenesis

Pollination, pollen tube growth, and fertilization independently contribute to fruit set and development in tomato

In flowering plants, pollination, pollen tube growth, and fertilization are regarded as the first hierarchical processes of producing offspring. However, their independent contributions to fruit set and development remain unclear. In this study, we examined the effect of three different types of pollen, intact pollen (IP), soft X-ray-treated pollen (XP) and dead pollen (DP), on pollen tube growth, fruit development and gene expression in “Micro-Tom” tomato. Normal germination and pollen tube growth were observed in flowers pollinated with IP; pollen tubes started to penetrate the ovary at 9 h after pollination, and full penetration was achieved after 24 h (IP24h), resulting in ~94% fruit set. At earlier time points (3 and 6 h after pollination; IP3h and IP6h, respectively), pollen tubes were still in the style, and no fruit set was observed. Flowers pollinated with XP followed by style removal after 24 h (XP24h) also demonstrated regular pollen tubes and produced parthenocarpic fruits with ~78% fruit set. As expected, DP could not germinate and failed to activate fruit formation. Histological analysis of the ovary at 2 days after anthesis (DAA) revealed that IP and XP comparably increased cell layers and cell size; however, mature fruits derived from XP were significantly smaller than those derived from IP. Furthermore, there was a high correlation between seed number and fruit size in fruit derived from IP, illustrating the crucial role of fertilization in the latter stages of fruit development. RNA-Seq analysis was carried out in ovaries derived from IP6h, IP24h, XP24h and DP24h in comparison with emasculated and unpollinated ovaries (E) at 2 DAA. The results revealed that 65 genes were differentially expressed (DE) in IP6h ovaries; these genes were closely associated with cell cycle dormancy release pathways. Conversely, 5062 and 4383 DE genes were obtained in IP24h and XP24h ovaries, respectively; top enriched terms were mostly associated with cell division and expansion in addition to the ‘plant hormone signal transduction’ pathway. These findings indicate that full penetration of pollen tubes can initiate fruit set and development independently of fertilization, most likely by activating the expression of genes regulating cell division and expansion

Involvement of vacuolar processing enzyme SlVPE5 in post-transcriptional process of invertase in sucrose accumulation in tomato

Enhancing the flavor of fruits plays a fundamental role in improving fruit quality, and volatile compositions as well as acid and sugar accumulation are significant factors that have an impact on the acceptability of sensory responses by human beings. Vacuoles in plants not only function as cell compartments that store amino acids, sugars and other metabolites but also act as lytic organelles where vacuolar proteins are post-translationally processed into mature forms or degraded by the action of vacuolar processing enzyme (VPE). We have previously characterized VPE genes (SlVPE1-5) during fruit development in tomato and discovered that the VPE enzyme activity negatively interfered with sugar accumulation in mature fruits. Comparative proteomic analysis demonstrated that acid invertase was one of the molecular targets of SlVPE5, which is involved in the hydrolysis of sucrose. This study also showed that decreased VPE enzyme activity due to suppression of SlVPE5 by RNAi strategy (RNAi-SlVPE5) accompanied with decreased enzyme activity of acid invertase. Further, we identified the enzyme activity of acid invertase was not well correlated with mRNA levels in the RNAi-SlVPE5 line. These results suggest that SlVPE5 regulates post-transcriptional processing through de novo synthesis of the acid invertase protein to suppress enzyme activity, thereby eventually ensuring sucrose hydrolysis

Genome-wide identification of pistil-specific genes expressed during fruit set initiation in tomato (Solanum lycopersicum)

Fruit set involves the developmental transition of an unfertilized quiescent ovary in the pistil into a fruit. While fruit set is known to involve the activation of signals (including various plant hormones) in the ovary, many biological aspects of this process remain elusive. To further expand our understanding of this process, we identified genes that are specifically expressed in tomato (Solanum lycopersicum L.) pistils during fruit set through comprehensive RNA-seq-based transcriptome analysis using 17 different tissues including pistils at six different developmental stages. First, we identified 532 candidate genes that are preferentially expressed in the pistil based on their tissue-specific expression profiles. Next, we compared our RNA-seq data with publically available transcriptome data, further refining the candidate genes that are specifically expressed within the pistil. As a result, 108 pistil-specific genes were identified, including several transcription factor genes that function in reproductive development. We also identified genes encoding hormone-like peptides with a secretion signal and cysteine-rich residues that are conserved among some Solanaceae species, suggesting that peptide hormones may function as signaling molecules during fruit set initiation. This study provides important information about pistil-specific genes, which may play specific roles in regulating pistil development in relation to fruit set

Suppression of γ-Aminobutyric Acid (GABA) Transaminases Induces Prominent GABA Accumulation, Dwarfism and Infertility in the Tomato (Solanum lycopersicum L.)

Tomatoes accumulate γ-aminobutyric acid (GABA) at high levels in the immature fruits. GABA is rapidly converted to succinate during fruit ripening through the activities of GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH). Although three genes encoding GABA-T and both pyruvate- and α-ketoglutarate-dependent GABA-T activities have been detected in tomato fruits, the mechanism underlying the GABA-T-mediated conversion of GABA has not been fully understood. In this work, we conducted loss-of-function analyses utilizing RNA interference (RNAi) transgenic plants with suppressed pyruvate- and glyoxylate-dependent GABA-T gene expression to clarify which GABA-T isoforms are essential for its function. The RNAi plants with suppressed SlGABA-T gene expression, particularly SlGABA-T1, showed severe dwarfism and infertility. SlGABA-T1 expression was inversely associated with GABA levels in the fruit at the red ripe stage. The GABA contents in 35S::SlGABA-T1RNAi lines were 1.3–2.0 times and 6.8–9.2 times higher in mature green and red ripe fruits, respectively, than the contents in wild-type fruits. In addition, SlGABA-T1 expression was strongly suppressed in the GABA-accumulating lines. These results indicate that pyruvate- and glyoxylate-dependent GABA-T is the essential isoform for GABA metabolism in tomato plants and that GABA-T1 primarily contributes to GABA reduction in the ripening fruits

Improvement of the transient expression system for production of recombinant proteins in plants

An efficient and high yielding expression system is required to produce recombinant proteins. Furthermore, the transient expression system can be used to identify the localization of proteins in plant cells. In this study, we demonstrated that combination of a geminiviral replication and a double terminator dramatically enhanced the transient protein expression level in plants. The GFP protein was expressed transiently in lettuce, Nicotiana benthamiana, tomatoes, eggplants, hot peppers, melons, and orchids with agroinfiltration. Compared to a single terminator, a double terminator enhanced the expression level. A heat shock protein terminator combined with an extensin terminator resulted in the highest protein expression. Transiently expressed GFP was confirmed by immunoblot analysis with anti-GFP antibodies. Quantitative analysis revealed that the geminiviral vector with a double terminator resulted in the expression of at least 3.7 mg/g fresh weight of GFP in Nicotiana benthamiana, approximately 2-fold that of the geminiviral vector with a single terminator. These results indicated that combination of the geminiviral replication and a double terminator is a useful tool for transient expression of the gene of interest in plant cells

Examining the Role of Low Temperature in Satsuma Mandarin Fruit Peel Degreening via Comparative Physiological and Transcriptomic Analysis

Peel degreening is the most conspicuous aspect of fruit ripening in many citrus fruits because of its importance for marketability. In this study, peel degreening in response to propylene (an ethylene analog) and at varying storage temperatures was characterized in Satsuma mandarin (Citrus unshiu Marc.) fruit. Propylene treatment triggered rapid peel degreening (within 4-6 days), indicated by an increase in the citrus color index (CCI) and chlorophyll loss. Peel degreening was also observed in fruit at 10 degrees C and 15 degrees C after 28-42 days, with gradual CCI increase and chlorophyll reduction. However, fruit at 5 degrees C, 20 degrees C, and 25 degrees C remained green, and no substantial changes in peel CCI and chlorophyll content were recorded during the 42-day storage duration. The transcriptomes of peels of fruit treated with propylene for 4 days and those stored at varying temperatures for 28 days were then analyzed by RNA-Seq. We identified three categories of differentially expressed genes that were regulated by (i) propylene (and by analogy, ethylene) alone, (ii) low temperature (5 degrees C, 10 degrees C, or 15 degrees C vs. 25 degrees C) alone, and (iii) either propylene or low temperature. Gene-encoding proteins associated with chlorophyll degradation (such as CuSGR1, CuNOL, CuACD2, CuCAB2, and CuLHCB2) and a transcription factor (CuERF114) were differentially expressed by propylene or low temperature. To further examine temperature-induced pathways, we also monitored gene expression during on-tree fruit maturation vs. postharvest. The onset of on-tree peel degreening coincided with autumnal drops in field temperatures, and it was accompanied by differential expression of low temperature-regulated genes. On the contrary, genes that were exclusively regulated by propylene (such as CuCOPT1 and CuPOX-A2) displayed insignificant expression changes during on-tree peel degreening. These findings indicate that low temperatures could be involved in the fruit ripening-related peel degreening independently of ethylene