CaGLK2 regulates natural variation of chlorophyll content and fruit color in pepper fruit.

KEY MESSAGE: We provide multiple evidences that CaGLK2 underlies a quantitative trait locus controlling natural variation in chlorophyll content and immature fruit color of pepper via modulating chloroplast compartment size. Pepper fruit quality is attributed to a variety of traits, affecting visual appearance, flavor, chemical composition and nutritional value. Among the quality traits, fruit color is of primary importance because the pigments that confer color are associated with nutrition, health and flavor. Although gene models have been proposed for qualitative aspects of fruit color, large natural variation in quantitative pigment content and fruit color exists in pepper. However, its genetic basis is largely unknown which hampers its utilization for plant improvement. We studied the role of GLK2, a GOLDEN2-like transcription factor that regulates chloroplast development in controlling natural variation for chlorophyll content and immature fruit color of pepper. The role of GLK2 in regulating fruit development has been studied previously in tomato using ectopic expression and the uniform ripening mutant analyses. However, pepper provides a unique opportunity to further study the function of this gene because of the wide natural variation of fruit colors in this species. Segregation, sequencing and expression analyses indicated that pepper GLK2 (CaGLK2) corresponds to the recently reported pc10 QTL that controls chloroplast development and chlorophyll content in pepper. CaGLK2 exerts its effect on chloroplast compartment size predominantly during immature fruit development. We show that the genetic background, sequence variation and expression pattern confer a complex and multi-level regulation of CaGLK2 and fruit color in Capsicum. The positive effect on fruit quality predominantly at the green stage conferred by CaGLK2 can be utilized to breed green pepper varieties with improved nutritional values and taste.

pc8.1, a major QTL for pigment content in pepper fruit, is associated with variation in plastid compartment size.

Studies on the genetic control of pigment content in pepper fruit have focused mainly on monogenic mutations leading to changes in fruit color. In addition to the qualitative variation in fruit color, quantitative variation in pigment content and color intensity exists in pepper giving rise to a range of color intensities. However, the genetic basis for this variation is poorly understood, hindering the development of peppers that are rich in these beneficial compounds. In this paper, quantitative variation in pigment content was studied in a cross between a dark-green Capsicum annuum pepper and a light-green C. chinense pepper. Two major pigment content QTLs that control chlorophyll content were identified, pc8.1 and pc10.1. The major QTL pc8.1, also affected carotenoid content in the ripe fruit. However, additional analyses in subsequent generations did not reveal a consistent effect of this QTL on carotenoid content in ripe fruit. Confocal microscopy analyses of green immature fruits of the parents and of near-isogenic lines for pc8.1 indicated that the QTL exerts its effect via increasing chloroplast compartment size in the dark-green genotypes, predominantly in a fruit-specific manner. Metabolic analyses indicated that in addition to chlorophyll, chloroplast-associated tocopherols and carotenoids are also elevated. Future identification of the genes controlling pigment content QTLs in pepper will provide a better understanding of this important trait and new opportunities for breeding peppers and other Solanaceae species with enhanced nutritional value.

Dual N- and C-terminal processing of citrus chlorophyllase precursor within the plastid membranes leads to the mature enzyme.

Chl, the central player in harvesting light energy for photosynthesis, is enzymatically degraded during natural turnover, leaf senescence, fruit ripening or following biotic/abiotic stress induction. The photodynamic properties of Chl and its metabolites call for tight regulation of the catabolic pathway enzymes to avoid accumulation of intermediate breakdown products. Chlorophyllase, the Chl dephytilation enzyme, was previously demonstrated to be an initiator of Chl breakdown when transcriptionally induced to be expressed during ethylene-induced citrus fruit color break or when heterologously expressed in different plant systems. Citrus chlorophyllase was previously shown to be translated as a precursor protein, which is subsequently post-translationally processed to a mature form. We demonstrate that maturation of citrus chlorophyllase involves dual N- and C-terminal processing which appear to be rate-limiting post-translational events when chlorophyllase expression levels are high. The chlorophyllase precursor and intermediate forms were shown to be of transient nature, while the mature form accumulates over time, suggesting that processing may be involved in post-translational regulation of enzyme in vivo function. This notion is further supported by the finding that neither N- nor C-terminal processed domains are essential for chloroplast targeting of the enzyme, and that both processing events occur within the chloroplast membranes. Studies on the processing of chlorophyllase versions truncated at the N- or C-termini or mutated to abolish C-terminal processing suggest that each of the processing events is independent. Dual N- and C-terminal processing, not involving an organellar targeting signal, has rarely been documented in plants and is unique for a plastid protein.

The NAC-domain transcription factor GOBLET specifies leaflet boundaries in compound tomato leaves.

Leaves are formed at the flanks of the shoot apical meristem (SAM) and develop into a variety of forms. In tomato, prolonged leaf patterning enables the elaboration of compound leaves by reiterative initiation of leaflets with lobed margins. In goblet (gob) loss-of-function mutants, primary leaflets are often fused, secondary leaflets and marginal serrations are absent, and SAMs often terminate precociously. We show that GOB encodes a NAC-domain transcription factor expressed in narrow stripes at the leaf margins, flanking the distal side of future leaflet primordia, and at the boundaries between the SAM and leaf primordia. Leaf-specific overexpression of the microRNA miR164, a negative regulator of GOB-like genes, also leads to loss of secondary-leaflet initiation and to smooth leaflet margins. Plants carrying a dominant gob allele with an intact ORF but disrupted miR164 binding site produce more cotyledons and floral organs, have split SAMs and, surprisingly, simpler leaves. Overexpression of a form of GOB with an altered miR164 binding site in leaf primordia leads to delayed leaflet maturation, frequent, improperly timed and spaced initiation events, and a simple mature leaflet form owing to secondary-leaflet fusion. miR164 also affects leaflet separation in Cardamine hirsuta, a Brassicaceae species with complex leaves. Genetic and molecular analyses suggest that GOB expression is intact in the simplified leaves of entire tomato mutants, which have a defect in a putative repressor of auxin responses. Our results show that GOB marks leaflet boundaries and that its accurate spatial, temporal and quantitative activity affects leaf elaboration in a context-dependent manner.

Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato.

Plant leaves show pronounced plasticity of size and form. In the classical, partially dominant mutation Lanceolate (La), the large compound leaves of tomato (Solanum lycopersicum) are converted into small simple ones. We show that LA encodes a transcription factor from the TCP family containing an miR319-binding site. Five independent La isolates are gain-of-function alleles that result from point mutations within the miR319-binding site and confer partial resistance of the La transcripts to microRNA (miRNA)-directed inhibition. The reduced sensitivity to miRNA regulation leads to elevated LA expression in very young La leaf primordia and to precocious differentiation of leaf margins. In contrast, downregulation of several LA-like genes using ectopic expression of miR319 resulted in larger leaflets and continuous growth of leaf margins. Our results imply that regulation of LA by miR319 defines a flexible window of morphogenetic competence along the developing leaf margin that is required for leaf elaboration.

Meristem maintenance and compound-leaf patterning utilize common genetic mechanisms in tomato.

Balancing shoot apical meristem (SAM) maintenance and organ formation from its flanks is essential for proper plant growth and development and for the flexibility of organ production in response to internal and external cues. Leaves are formed at the SAM flanks and display a wide variability in size and form. Tomato (Solanum lycopersicum) leaves are compound with lobed margins. We exploited 18 recessive tomato mutants, representing four distinct phenotypic classes and six complementation groups, to track the genetic mechanisms involved in meristem function and compound-leaf patterning in tomato. In goblet (gob) mutants, the SAM terminates following cotyledon production, but occasionally partially recovers and produces simple leaves. expelled shoot (exp) meristems terminate after the production of several leaves, and these leaves show a reduced level of compoundness. short pedicel (spd) mutants are bushy, with impaired meristem structure, compact inflorescences, short pedicels and less compound leaves. In multi drop (mud) mutants, the leaves are more compound and the SAM tends to divide into two active meristems after the production of a few leaves. The range of leaf-compoundness phenotypes observed in these mutants suggests that compound-leaf patterning involves an array of genetic factors, which act successively to elaborate leaf shape. Furthermore, the results indicate that similar mechanisms underlie SAM activity and compound-leaf patterning in tomato.