Alternative transcription and feedback regulation suggest that SlIDI1 is involved in tomato carotenoid synthesis in a complex way.

Carotenoid pigments confer photoprotection and visual attraction and serve as precursors for many important signaling molecules. Herein, the orange-fruited phenotype of a tomato elite inbred line resulting from sharply reduced carotenoid levels and an increased β-carotene-to-lycopene ratio in fruit was shown to be controlled by a single recessive gene, oft3. BSA-Seq combined with fine mapping delimited the oft3 gene to a 71.23 kb interval on chromosome 4, including eight genes. Finally, the oft3 candidate gene SlIDI1, harboring a 116 bp deletion mutation, was identified by genome sequence analysis. Further functional complementation and CRISPR–Cas9 knockout experiments confirmed that SlIDI1 was the gene underlying the oft3 locus. qRT–PCR analysis revealed that the expression of SlIDI1 was highest in flowers and fruit and increased with fruit ripening or flower maturation. SlIDI1 simultaneously produced long and short transcripts by alternative transcription initiation and alternative splicing. Green fluorescent protein fusion expression revealed that the long isoform was mainly localized in plastids and that an N-terminal 59-amino acid extension sequence was responsible for plastid targeting. Short transcripts were identified in leaves and fruit by 5’ RACE and in fruit by 3’ RACE, which produced corresponding proteins lacking transit peptides and/or putative peroxisome targeting sequences, respectively. In SlIDI1 mutant fruit, SlBCH1 transcription involved in β-carotenoid catabolism was obviously suppressed, which may be responsible for the higher β-carotene-to-lycopene ratio and suggested potential feedback regulatory mechanisms involved in carotenoid pathway flux.

Plastidial wax ester biosynthesis as a tool to synthesize shorter and more saturated wax esters

Background Wax esters (WE) are neutral lipids that consist of a fatty alcohol esterified to a fatty acid. WE are valuable feedstocks in industry for producing lubricants, coatings, and cosmetics. They can be produced chemically from fossil fuel or plant-derived triacylglycerol. As fossil fuel resources are finite, the synthesis of WE in transgenic plants may serve as an alternative source. As chain length and desaturation of the alcohol and acyl moieties determine the physicochemical properties of WE and their field of application, tightly controlled and tailor-made WE synthesis in plants would be a sustainable, beneficial, and valuable commodity. Here, we report the expression of ten combinations of WE producing transgenes in Arabidopsis thaliana. In order to study their suitability for WE production in planta, we analyzed WE amount and composition in the transgenic plants. Results The transgenes consisted of different combinations of a FATTY ACYL-COA/ACP REDUCTASE (FAR) and two WAX SYNTHASES/ACYL-COA:DIACYLGLYCEROL O-ACYLTRANSFERASES (WSD), namely WSD2 and WSD5 from the bacterium Marinobacter aquaeoleoi. We generated constructs with and without plastidial transit peptides to access distinct alcohol and acyl substrate pools within A. thaliana cells. We observed WE formation with plastid and cytosol-localized FAR and WSD in seeds. A comparative WE analysis revealed the production of shorter and more saturated WE by plastid-localized WE biosynthesis compared to cytosolic WE synthesis. Conclusions A shift of WE formation into seed plastids is a suitable approach for tailor-made WE production and can be used to synthesize WE that are mainly derived from mid- and long-chain saturated and monounsaturated substrates.

Chloroplast transit peptides often require downstream unstructured sequence in Chlamydomonas reinhardtii

The N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for nine out of ten tested Chlamydomonas chloroplast transit peptides, additional sequence past the cleavage site is required to enable chloroplast targeting. Using replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini.

Functional Organization of Sequence Motifs in Diverse Transit Peptides of Chloroplast Proteins

Although the chloroplasts in plants are characterized by an inherent genome, the chloroplast proteome is composed of proteins encoded by not only the chloroplast genome but also the nuclear genome. Nuclear-encoded chloroplast proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the chloroplasts. In the latter process, an N-terminal cleavable transit peptide serves as a targeting signal required for the import of nuclear-encoded chloroplast interior proteins. This import process is mediated via an interaction between the sequence motifs in transit peptides and the components of the TOC/TIC (translocon at the outer/inner envelope of chloroplasts) translocons. Despite a considerable diversity in primary structures, several common features have been identified among transit peptides, including N-terminal moderate hydrophobicity, multiple proline residues dispersed throughout the transit peptide, preferential usage of basic residues over acidic residues, and an absence of N-terminal arginine residues. In this review, we will recapitulate and discuss recent progress in our current understanding of the functional organization of sequence elements commonly present in diverse transit peptides, which are essential for the multi-step import of chloroplast proteins.

Plastidial Wax Ester Biosynthesis As A Tool To Synthesize Shorter And More Saturated Wax Esters

Background: Wax esters (WE) are neutral lipids that consist of a fatty alcohol esterified to a fatty acid. WE are valuable feedstocks in industry for producing lubricants, coatings and cosmetics. They can be produced chemically from fossil fuel or plant derived triacylglycerol. As fossil fuel resources are finite, the synthesis of WE in transgenic plants may serve as an alternative source. As chain length and desaturation degree of the alcohol and acyl moieties determine the physicochemical properties of WE and their field of application, a tightly controlled tailor-made WE synthesis in plants is aimed for. Here, we report the generation of ten combinations of WE producing transgenes expressed in Arabidopsis thaliana. In order to study their suitability for WE production in planta, we analyzed WE amount and synthesized WE species in the transgenic plants.Results: The transgenes consisted of different combinations of a fatty acyl-CoA/ACP reductase (FAR) and two wax synthases/acyl-CoA:diacylglycerol O-acyltransferases (WSD) namely WSD2 and WSD5 from the bacterium Marinobacter aquaeoleoi. We generated constructs with and without plastidial transit peptides to access diverse alcohol and acyl substrate pools within A. thaliana. We observed WE formation with plastid and cytosol-localized FAR and WSD in seeds. A comparative WE analysis revealed the production of shorter and more saturated WE by plastid-localized WE biosynthesis compared to cytosolic WE synthesis.Conclusions: A shift of WE formation into seed plastids is a suitable approach for tailor-made WE production and can be used to synthesize WE mainly derived from mid and long chain saturated and monounsaturated substrates.

A molecular toolkit for the green seaweed Ulva mutabilis

The green seaweed Ulva is an ecologically-important marine primary producer as well as a promising cash crop cultivated for multiple uses. Despite its importance, several molecular tools are still needed to better understand seaweed biology. Here, we report the development of a flexible and modular molecular cloning toolkit for the green seaweed Ulva mutabilis based on a Golden Gate cloning system. The toolkit presently contains 125 entry vectors, 26 destination vectors and 107 functionally validated expression vectors. We demonstrate the importance of endogenous regulatory sequences for transgene expression and characterize three endogenous promoters suitable to drive transgene expression. We describe two vector architectures to express transgenes via two expression cassettes or a bicistronic approach. The majority of selected transformants (50-80%) consistently give clear visual transgene expression. Furthermore, we made different marker lines for intracellular compartments after evaluating 13 transit peptides and 11 tagged endogenous Ulva genes. Our molecular toolkit enables the study of Ulva gain-of-function lines and paves the way for gene characterization and large-scale functional genomics studies in a green seaweed.

TIC236 gain-of-function mutations unveil the link between plastid division and plastid protein import

The chloroplast translocons TOC75 and TIC236 are homologs of the bacterial translocation and assembly module (Tam) A and TamB involved in protein export. Here, we unveil a TIC236-allied component, the chloroplast outer membrane protein CRUMPLED LEAF (CRL), absence of which impairs plastid division and induces autoimmune responses in Arabidopsis thaliana. A forward genetic screen aimed at finding crl suppressors revealed multiple TIC236 gain-of-function mutations (TIC236GFs). Despite the low sequence identity between TIC236 and bacterial TamB, each mutated TIC236GF residue is conserved in TamB. Consistently, a tic236-knockdown mutant exhibited multiple lesion phenotypes similar to crl, indicating a shared functionality of CRL and TIC236. Ensuing reverse genetic analyses revealed genetic interaction between CRL and SP1, a RING-type ubiquitin E3 ligase, as well as with the plastid protease FTSH11, which function in TOC and TIC protein turnover, respectively. Loss of either SP1 or FTSH11 rescued crl mutant phenotypes to varying degrees due to increased translocon levels. Consistent with impaired plastid division exhibited by both crl and tic236-knockdown mutants, CRL interacts with the transit peptides of proteins essential in plastid division, and TIC236GF mutant proteins reinforce their import via increased TIC236 stability. Overall, our data shed new light on the links between plastid division, plant stress response and plastid protein import. We have also isolated and characterized the first GF mutants exhibiting increased protein import efficiency, which may inspire chloroplast engineering for agricultural advancement.

The ten amino acids of the oxygen-evolving enhancer of tobacco is sufficient as the peptide residues for protein transport to the chloroplast thylakoid

Key message The thylakoid transit peptide of tobacco oxygen-evolving enhancer protein contains a minimal ten amino acid sequences for thylakoid lumen transports. This ten amino acids do not contain twin-arginine, which is required for typical chloroplast lumen translocation. Abstract Chloroplasts are intracellular organelles responsible for photosynthesis to produce organic carbon for all organisms. Numerous proteins must be transported from the cytosol to chloroplasts to support photosynthesis. This transport is facilitated by chloroplast transit peptides (TPs). Four chloroplast thylakoid lumen TPs were isolated from Nicotiana tabacum and were functionally analyzed as thylakoid lumen TPs. Typical chloroplast stroma-transit peptides and thylakoid lumen transit peptides (tTPs) are found in N. tabacum transit peptides (NtTPs) and the functions of these peptides are confirmed with TP–GFP fusion proteins under fluorescence microscopy and chloroplast fractionation, followed by Western blot analysis. During the functional analysis of tTPs, we uncovered the minimum 10 amino acid sequence is sufficient for thylakoid lumen transport. These ten amino acids can efficiently translocate GFP protein, even if they do not contain the twin-arginine residues required for the twin-arginine translocation (Tat) pathway, which is a typical thylakoid lumen transport. Further, thylakoid lumen transporting processes through the Tat pathway was examined by analyzing tTP sequence functions and we demonstrate that the importance of hydrophobic core for the tTP cleavage and target protein translocation.

A molecular toolkit for the green seaweed Ulva mutabilis

The green seaweed Ulva is an ecologically-important marine primary producer as well as a promising cash crop cultivated for multiple uses. Despite its importance, several molecular tools are still needed to better understand seaweed biology. Here, we report the development of a flexible and modular molecular cloning toolkit for the green seaweed Ulva mutabilis based on a Golden Gate cloning system. The toolkit presently contains 125 entry vectors, 26 destination vectors and 107 functionally validated expression vectors. We demonstrate the importance of endogenous regulatory sequences for transgene expression and characterize three endogenous promoters suitable to drive transgene expression. We describe two vector architectures to express transgenes via two expression cassettes or a bicistronic approach. The majority of selected transformants (50-80%) consistently give clear visual transgene expression. Furthermore, we made different marker lines for intracellular compartments after evaluating 13 transit peptides and 11 tagged endogenous Ulva genes. Our molecular toolkit enables the study of Ulva gain-of-function lines and paves the way for gene characterization and large-scale functional genomics studies in a green seaweed.

Structural and functional features of phytoene synthase isoforms PSY1 and PSY2 in pepper Capsicum annuum L. cultivars

The fruits of various pepper cultivars are characterized by a different colour, which is determined by the pigment ratio; carotenoids dominate in ripe fruits, while chlorophylls, in immature fruits. A key regulator of carotenoid biosynthesis is the phytoene synthase encoded by the PSY gene. The Capsicum annuum genome contains two isoforms of this enzyme, localized in leaf (PSY2) and fruit (PSY1) plastids. In this work, the complete PSY1 and PSY2 genes were identified in nine C. annuum cultivars, which differ in ripe fruit colour. PSY1 and PSY2 sequence variability was 2.43 % (69 SNPs) and 1.21 % (36 SNPs). The most variable were PSY1 proteins of the cultivars ‘Maria’ (red-fruited) and ‘Sladkij shokolad’ (red-brown-fruited). All identified PSY1 and PSY2 homologs contained the phytoene synthase domain HH-IPPS and the transit peptide. In the PSY1 and PSY2 HH-IPPS domains, functionally significant sites were determined. For all accessions studied, the active sites (YAKTF and RAYV), aspartate-rich substrate-Mg2+-binding sites (DELVD and DVGED), and other functional residues were shown to be conserved. Transit peptides were more variable, and their similarity in the PSY1 and PSY2 proteins did not exceed 78.68 %. According to the biochemical data obtained, the largest amounts of chlorophylls and carotenoids across the cultivars studied were detected in immature and ripe fruits of the cv. ‘Sladkij shokolad’ and ‘Shokoladnyj’. Also, ripe fruits of the cv. ‘Nesozrevayuschij’ (green-fruited) were marked by significant chlorophyll content, but a minimum of carotenoids. The PSY1 and PSY2 expression patterns were determined in the fruit pericarp at three ripening stages in ‘Zheltyj buket’, ‘Sladkij shokolad’, ‘Karmin’ and ‘Nesozrevayuschij’, which have different ripe fruit colours: yellow, red-brown, dark red and green, respectively. In the leaves of the cultivars studied, PSY1 expression levels varied significantly. All cultivars were characterized by increased PSY1 transcription as the fruit ripened; the maximum transcription level was found in the ripe fruit of ‘Sladkij shokolad’, and the lowest, in ‘Nesozrevayuschij’. PSY2 transcripts were detected not only in the leaves and immature fruits, but also in ripe fruits. Assessment of a possible correlation of PSY1 and PSY2 transcription with carotenoid and chlorophyll content revealed a direct relationship between PSY1 expression level and carotenoid pigmentation during fruit ripening. It has been suggested that the absence of a typical pericarp pigmentation pattern in ‘Nesozrevayuschij’ may be associated with impaired chromoplast formation.