Induced point mutations in the phytoene synthase 1 gene cause differences in carotenoid content during tomato fruit ripening

In tomato, carotenoids are important with regard to major breeding traits such as fruit colour and human health. The enzyme phytoene synthase (PSY1) directs metabolic flux towards carotenoid synthesis. Through TILLING (Targeting Induced Local Lesions IN Genomes), we have identified two point mutations in the Psy1 gene. The first mutation is a knockout allele (W180*) and the second mutation leads to an amino acid substitution (P192L). Plants carrying the Psy1 knockout allele show fruit with a yellow flesh colour similar to the r, r mutant, with no further change in colour during ripening. In the line with P192L substitution, fruit remain yellow until 3 days post-breaker and eventually turn red. Metabolite profiling verified the absence of carotenoids in the W180* line and thereby confirms that PSY1 is the only enzyme introducing substrate into the carotenoid pathway in ripening fruit. More subtle effects on carotenoid accumulation were observed in the P192L line with a delay in lycopene and β-carotene accumulation clearly linked to a very slow synthesis of phytoene. The observation of lutein degradation with ripening in both lines showed that lutein and its precursors are still synthesised in ripening fruit. Gene expression analysis of key genes involved in carotenoid biosynthesis revealed that expression levels of genes in the pathway are not feedback-regulated by low levels or absence of carotenoid compounds. Furthermore, protein secondary structure modelling indicated that the P192L mutation affects PSY1 activity through misfolding, leading to the low phytoene accumulation.     

Regulation of carotenoid biosynthesis during tomato development.

Phytoene synthase (Psy) and phytoene desaturase (Pds) are the first dedicated enzymes of the plant carotenoid biosynthesis pathway. We report here the organ-specific and temporal expression of PDS and PSY in tomato plants. Light increases the carotenoid content of seedlings but has little effect on PDS and PSY expression. Expression of both genes is induced in seedlings of the phytoene-accumulating mutant ghost and in wild-type seedlings treated with the Pds inhibitor norflurazon. Roots, which contain the lowest levels of carotenoids in the plant, have also the lowest levels of PDS and PSY expression. In flowers, expression of both genes and carotenoid content are higher in petals and anthers than in sepals and carpels. During flower development, expression of both PDS and PSY increases more than 10-fold immediately before anthesis. During fruit development, PSY expression increases more than 20-fold, but PDS expression increases less than threefold. We concluded that PSY and PDS are differentially regulated by stress and developmental mechanisms that control carotenoid biosynthesis in leaves, flowers, and fruits. We also report that PDS maps to chromosome 3, and thus it does not correspond to the GHOST locus, which maps to chromosome 11.     

Arachidonic Acid Alters Tomato HMG Expression and Fruit Growth and Induces 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase-Independent Lycopene Accumulation

Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, we manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Our results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.     

Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-D-xylulose 5-phosphate synthase

Plant isoprenoids represent a heterogeneous group of compounds which play essential roles not only in growth and development, but also in the interaction of plants with their environment. Higher plants contain two pathways for the biosynthesis of isoprenoids: the mevalonate pathway, located in the cytosol/endoplasmic reticulum, and the recently discovered mevalonate-independent pathway (Rohmer pathway), located in the plastids. In order to evaluate the function of the Rohmer pathway in the regulation of the synthesis of plastidial isoprenoids, we have isolated a tomato cDNA encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the first enzyme of the pathway. We demonstrate in vivo activity and plastid targeting of plant DXS. Expression analysis of the tomato DXS gene indicates developmental and organ-specific regulation of mRNA accumulation and a strong correlation with carotenoid synthesis during fruit development. 1-Deoxy-D-xylulose feeding experiments, together with expression analysis of DXS and PSY1 (encoding the fruit-specific isoform of phytoene synthase) in wild-type and yellow flesh mutant fruits, indicate that DXS catalyses the first potentially regulatory step in carotenoid biosynthesis during early fruit ripening. Our results change the current view that PSY1 is the only regulatory enzyme in tomato fruit carotenogenesis, and point towards a coordinated role of both DXS and PSY1 in the control of fruit carotenoid synthesis.     

Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity

Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima’D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.     

The sub-cellular localisation of the potato (Solanum tuberosum L.) carotenoid biosynthetic enzymes, CrtRb2 and PSY2

Carotenoids are isoprenoids with important biological roles both for plants and animals. The yellow flesh colour of potato (Solanum tuberosum L.) tubers is a quality trait dependent on the types and levels of carotenoids that accumulate. The carotenoid biosynthetic pathway is well characterised, facilitating the successful engineering of carotenoid content in numerous crops including potato. However, a clear understanding concerning the factors regulating carotenoid accumulation and localisation in plant storage organs, such as tubers, is lacking. In the present study, the localisation of key carotenoid biosynthetic enzymes was investigated, as one of the unexplored factors that could influence the accumulation of carotenoids in potato tubers. Stable transgenic potato plants were generated by over-expressing β-CAROTENE HYDROXYLASE 2 (CrtRb2) and PHYTOENE SYNTHASE 2 (PSY2) genes, fused to red fluorescent protein (RFP). Gene expression and carotenoid levels were both significantly increased, confirming functionality of the fluorescently tagged proteins. Confocal microscopy studies revealed different sub-organellar localisations of CrtRb2-RFP and PSY2-RFP within amyloplasts. CrtRb2 was detected in small vesicular structures, inside amyloplasts, whereas PSY2 was localised in the stroma of amyloplasts. We conclude that it is important to consider the location of biosynthetic enzymes when engineering the carotenoid metabolic pathway in storage organs such as tubers.     

CRISPR/Cas9 editing of carotenoid genes in tomato

CRISPR/Cas9 technology is rapidly spreading as genome editing system in crop breeding. The efficacy of CRISPR/Cas9 in tomato was tested on Psy1 and CrtR-b2, two key genes of carotenoid biosynthesis. Carotenoids are plant secondary metabolites that must be present in the diet of higher animals because they exert irreplaceable functions in important physiological processes. Psy1 and CrtR-b2 were chosen because their impairment is easily detectable as a change of fruit or flower color. Two CRISPR/Cas9 constructs were designed to target neighboring sequences on the first exon of each gene. Thirty-four out of forty-nine (69%) transformed plants showed the expected loss-of-function phenotypes due to the editing of both alleles of a locus. However, by including the seven plants edited only at one of the two homologs and showing a normal phenotype, the editing rate reaches the 84%. Although none chimeric phenotype was observed, the cloning of target region amplified fragments revealed that in the 40% of analyzed DNA samples were present more than two alleles. As concerning the type of mutation, it was possible to identify 34 new different alleles across the four transformation experiments. The sequence characterization of the CRISPR/Cas9-induced mutations showed that the most frequent repair errors were the insertion and the deletion of one base. The results of this study prove that the CRISPRCas9 system can be an efficient and quick method for the generation of useful mutations in tomato to be implemented in breeding programs.     

Regulatory control of carotenoid accumulation in winter squash during storage

Main conclusion

Storage promotes carotenoid accumulation and converts amylochromoplasts into chromoplasts in winter squash. Such carotenoid enhancement is likely due to continuous biosynthesis along with reduced turnover and/or enhanced sequestration. Postharvest storage of fruits and vegetables is often required and frequently results in nutritional quality change. In this study, we investigated carotenoid storage plastids, carotenoid content, and its regulation during 3-month storage of winter squash butternut fruits. We showed that storage improved visual appearance of fruit flesh color from light to dark orange, and promoted continuous accumulation of carotenoids during the first 2-month storage. Such an increased carotenoid accumulation was found to be concomitant with starch breakdown, resulting in the conversion of amylochromoplasts into chromoplasts. The butternut fruits contained predominantly β-carotene, lutein, and violaxanthin. Increased ratios of β-carotene and violaxanthin to total carotenoids were noticed during the storage. Analysis of carotenoid metabolic gene expression and PSY protein level revealed a decreased expression of carotenogenic genes and PSY protein following the storage, indicating that the increased carotenoid level might not be due to increased biosynthesis. Instead, the increase likely resulted from a continuous biosynthesis with a possibly reduced turnover and/or enhanced sequestration, suggesting a complex regulation of carotenoid accumulation during fruit storage. This study provides important information to our understanding of carotenogenesis and its regulation during postharvest storage of fruits.     

Changes in the Alkaloid Content of Developing Fruits of Tomato (Lycopersicon esculentum Mill.): II. EFFECTS OF ARTIFICIAL ACCELERATION AND RETARDATION OF RIPENING

Fruit ripening in normal red-, orange- and yellow-fruited cultivarsof tomato was accelerated by treatment with Ethrel and suchfruits had lower tomatine levels than untreated controls. Fruitsin which ripening was retarded by incubating under reduced pressurehad higher alkaloid levels than controls. In each case, fruitripeness (as measured by pigmentation) showed a strong negativecorrelation with fruit tomatine. Ethrel treatment of fruitsof the non-ripening mutants nor and rin caused only a smalldevelopment of carotenoid pigment but markedly enhanced tomatinedisappearance. Pigmentation and tomatine were again negativelycorrelated although the quantitative relationship differed.Under normal circumstances, tomatine disappearance from tomatofruits-is apparently governed by the physiological (cf. chronological)age of the fruit rather than by its growth or ripening characteristicsalone. Key words: Tomato, Fruit ripening, Acceleration, Retardation, Tomatine     

西双版纳黄瓜Cs-Psy1基因的序列特征与表达分析

西双版纳黄瓜是我国特有的果肉橙黄色的黄瓜变种资源,不同种质间的β-胡萝卜素含量差异明显。PSY是胡萝卜素生物合成途径中的第1个限速酶。本文以西双版纳黄瓜为试材,分别克隆西双版纳黄瓜八氢番茄红素合成酶(Cs-PSY1)的DNA和c DNA序列,结果显示,DNA长2797 bp,包含5个内含子和6个外显子,c DNA序列长1385 bp,编码421个氨基酸。Psy1推测的氨基酸序列包含该家族的2个特征序列,保守性很高。该蛋白为不稳定蛋白,无明显疏水区,未预测到跨膜结构;系统进化分析结果显示,西双版纳黄瓜的Cs-PSY1蛋白与甜瓜的同源性较高;与栽培黄瓜深度测序材料"9930"和"GY14"的序列进行比较分析,结合115份黄瓜重测序结果,共发现5个SNP,其中2个位于起始密码子上游27 bp处和971 bp处,3个位于内含子区域。其中SNP4在重测序的19份西双版纳黄瓜中的突变率为100%,在96份栽培黄瓜中的特异性为5.3%。转录因子结合位点预测结果显示,在普通栽培黄瓜该位点处存在一个CTAG motif,在西双版纳黄瓜中该位点突变后则不存在该motif。利用实时荧光定量PCR技术分析Cs-Psy1的表达量变化趋势,结果表明,在黄瓜不同果实发育时期,该基因的表达量均呈现先上升后下降的趋势,在西双版纳黄瓜中表达量变化的差异明显,在授粉后50 d达到最大值,是果实发育初期表达量的8倍多,是同时期普通黄瓜的4倍多,而普通黄瓜表达量的总体变化相对平缓。西双版纳黄瓜果实内果皮的表达水平明显高于中果皮,最高相差约5倍,普通黄瓜差异不明显。从上述研究结果推测Psy1基因可能影响西双版纳黄瓜的β-胡萝卜素积累。     

A tomato gene expressed during fruit ripening encodes an enzyme of the carotenoid biosynthesis pathway.

In the initial stages of carotenoid biosynthesis in plants the enzyme phytoene synthase converts two molecules of geranylgeranyl diphosphate into phytoene, the first carotenoid of the pathway. We show here that a tomato (Lycopersicon esculentum) cDNA for a gene (Psy1) expressed during fruit ripening directs the in vitro synthesis of a 47-kDa protein which, upon import into isolated chloroplasts, is processed to a mature 42-kDa form. The imported protein is largely associated with membranes, but it can be easily solubilized by dilution or by treatment at high pH. A plasmid construct containing prokaryotic promoter and ribosome-binding sequences fused to the Psy1 cDNA complements the carotenoidless phenotype of a Rhodobacter capsulatus crtB mutant. We conclude that Psy1 encodes phytoene synthase and that this enzyme is a peripheral plastid membrane protein.     

Phosphatase Complex Pph3/Psy2 Is Involved in Regulation of Efficient Non-Homologous End-Joining Pathway in the Yeast Saccharomyces cerevisiae

One of the main mechanisms for double stranded DNA break (DSB) repair is through the non-homologous end-joining (NHEJ) pathway. Using plasmid and chromosomal repair assays, we showed that deletion mutant strains for interacting proteins Pph3p and Psy2p had reduced efficiencies in NHEJ. We further observed that this activity of Pph3p and Psy2p appeared linked to cell cycle Rad53p and Chk1p checkpoint proteins. Pph3/Psy2 is a phosphatase complex, which regulates recovery from the Rad53p DNA damage checkpoint. Overexpression of Chk1p checkpoint protein in a parallel pathway to Rad53p compensated for the deletion of PPH3 or PSY2 in a chromosomal repair assay. Double mutant strains Δpph3/Δchk1 and Δpsy2/Δchk1 showed additional reductions in the efficiency of plasmid repair, compared to both single deletions which is in agreement with the activity of Pph3p and Psy2p in a parallel pathway to Chk1p. Genetic interaction analyses also supported a role for Pph3p and Psy2p in DNA damage repair, the NHEJ pathway, as well as cell cycle progression. Collectively, we report that the activity of Pph3p and Psy2p further connects NHEJ repair to cell cycle progression.     

An increase of lycopene content in tomato fruit is associated with a novel Cyc-B allele isolated through TILLING technology

Tomato Cyc-B gene encodes a chromoplast-specific lycopene β-cyclase that converts lycopene to β-carotene during ripening of the fruit. By screening the tomato Red Setter mutant population with the TILLING method, we identified eight new alleles at the Cyc-B locus. Results of greenhouse phenotypic analysis revealed that the novel A949G Cyc-B allele produced modifications in the carotenoid profile and content of tomato petals and fruit. The cyc-b7 genotype, carrying the A949G Cyc-B allele, was therefore evaluated in an open field trial for standard agronomic traits as well as carotenoid content of the fruit. Results of the field trial confirmed that the induced A949G missense mutation favored the accumulation of lycopene in the fruit with no detrimental effects on the yield or on other agronomic and technological properties such as fruit firmness and Brix degree of fruit juice. On the basis of these results, it can be affirmed that the A949G Cyc-B allele constitutes a useful new genetic variant which can be used for improving carotenoid content in tomato fruit and for the development of new tomato commercial lines. Finally, the results presented here furthermore demonstrate that TILLING is a powerful methodology not only as a confirmatory system for gene functional analysis but also for selecting new gene variants useful for genetic improvement of important crops.