STAY-GREEN in orange: uncoupled functions in chlorophyll and carotenoid accumulation

How can we recognize the onset of fruit ripening? In many cases, color change from immature green to vivid red or orange indicates ripening and attracts seed-dispersing organisms at the right time. In many fruits, like tomato (Solanum lycopersicum) and pepper (Capsicum annuum), the change in color from green to red or orange is due to a sharp decrease in chlorophyll and an increase in carotenoid accumulation. Tomato has been intensively used as a model system of fruit ripening through which many important factors have been characterized (Chen et al., 2020).

Citruses are economically and nutritionally important crops with a global consumption of around 50 million tons every year (US Department of Agriculture, 2021). However, knowledge of the molecular regulation of citrus fruit ripening is limited. First, the direct application of knowledge from tomatoes is difficult because of the morphological difference in fruits. Citrus fruit is “hesperidia,” with sectioned pulp inside a separable leathery rind. The rind is composed of the outer exocarp (also called flavedo) and the pithy mesocarp (also called albedo; Figure 1). Chlorophyll accumulation in citrus is limited to the flavedo, in contrast to tomato, in which chlorophyll accumulates throughout the fruit pericarp tissues. Another challenge to understand the genetic basis of ripening in citrus is the complexity of citrus genomes. Domesticated citrus is interspecific hybrids with different degrees of admixture from progenitors. In sweet orange (Citrus sinensis), a backcross-like hybrid between pummelo and mandarin, about half of predicted protein-encoding genes are heterozygous with two different alleles (Xu et al., 2013).

Figure 1.

The regulatory model of fruit color by STAY-GREEN in citrus and tomato. Citrus and tomato have distinctive morphology in fruits. In citrus, chlorophyll accumulation is limited to the flavedo (exocarp), in contrast, chlorophyll accumulates throughout the pericarp tissues in tomato fruits. It has been revealed SGR is responsible for chlorophyll degradation in tomato. Zhu et al. (2021) find that there are two STAY-GREEN (SGR) alleles, SGRa and SGRb, in wild-type navel orange. Only SGRa is functional in inducing chlorophyll degradation. SGRb is a truncated version in both wild type and the mutant “Zong Cheng.” In “Zong Cheng,” the mutated version, SGRa^STOP, is not functional, leading to the accumulation of chlorophylls together with carotenoids, which results in brown-colored mature fruit. Meanwhile, both SGRa and SGRb can inhibit carotenoid biosynthesis through the direct interaction with PSY. SGRa may contribute more than SGRb in the repression of carotenoid accumulation since the expression level of SGRb is lower.

In this issue of Plant Physiology, Zhu et al. (2021) investigated the molecular basis of the brown flavedo mutant (Zong Cheng) in navel orange, a variety of sweet orange. Through this work, they discovered an uncoupling of chlorophyll and carotenoid pools in the flavedo during fruit ripening.

The Zong Cheng mutant was discovered from a spontaneous bud mutation of navel orange. Biochemical analysis revealed that while wild-type navel oranges only accumulate carotenoids in mature fruits, in Zong Cheng, both green-colored chlorophyll and higher levels of orange-colored carotenoid pigments accumulate and are responsible for the brown flavedo. Identifying the genetic basis is the first step to reveal the underlying regulatory mechanism but is difficult in long-lived woody perennial citrus with high heterozygosity because traditional map-based cloning is not suitable.

To uncover the genetic basis for the accumulation of chlorophyll in the flavedo in this mutant, Zhu et al. combined three sets of -omics data. The first two datasets came from differentially expressed genes between wild type and mutant and between green and orange sectors in chimeric mutant fruits by transcriptome analysis. The third dataset was generated by whole-genome sequencing of the mutant variety. The STAY-GREEN (SGR) gene was the most remarkable candidate gene identified from these three datasets. The authors found that, like many other loci in citrus, there are two different SGR alleles (SGRa and SGRb) present. While the SGRb allele is a truncated version with a 3′ deletion in both the wild-type and mutant varieties, in the mutant, the SGRa allele has a premature stop codon (SGRa^STOP), suggesting that this truncation is the cause of brown flavedo phenotype.

STAY-GREEN is a magnesium dechelatase that regulates chlorophyll degradation (Park et al., 2007). In tomato, a point mutation in SGR has been identified by positional cloning in the green-flesh (gf) mutant (Figure 1), which is responsible for the accumulation of chlorophyll in mature fruits (Barry et al., 2008). Based on this, the authors investigated the function of citrus SGRa, SGRb, and SGRa^STOP in chlorophyll degradation. Transient assays in leaves of Nicotiana benthamiana showed that only SGRa can induce chlorophyll degradation, which was further confirmed by in vitro enzyme assay (Figure 1). Because the SGRa allele is truncated in the Zong Cheng mutant, chlorophyll is not degraded in these fruits, leading to the brown color.

Both chlorophyll and carotenoid biosynthesis pathways are highly conserved across the plant kingdom, although the regulatory mechanism differs between different fruits. In tomato, it has been proposed that SGR can directly interact with PHYTOENE SYNTHASE (PSY), the rate-controlling enzyme for carotenoid biosynthesis, to limit carotenoid biosynthesis during fruit ripening (Luo et al., 2013). Therefore, the function of citrus SGR in the regulation of carotenoid biosynthesis was examined. Interestingly, both SGRa and SGRb, but not SGRa^STOP, can interact with PSY as revealed by bimolecular fluorescence complementation and yeast two-hybrid assays. Transformation of citrus callus showed that over-expressing SGRa and SGRb significantly repressed carotenoid accumulation, while the callus over-expressing SGRa^STOP still showed orange color with no difference from empty vector control. These results imply that both SGRa and SGRb can repress carotenoid biosynthesis through the interaction with PSY (Figure 1). Since the expression level of SGRb in fruit is lower than SGRa, these results are consistent with the observation that the mutant with loss-of-function in SGRa accumulates more carotenoids than wild type in flavedo.

In summary, Zhu et al. (2021) reveal the uncoupled regulatory mechanism of chlorophyll and carotenoid accumulation by SGR protein in citrus flavedo. Specifically, the SGRa allele both promotes chlorophyll degradation and represses carotenoid accumulation, whereas the SGRb allele only affects carotenoid accumulation. The discovery of this regulatory mechanism not only suggests potential targets for citrus fruit quality improvement in the future but also inspires functional gene discovery in other woody perennials of economic importance.

It would be interesting to further explore how the gene expression of SGRa and SGRb are coordinated during fruit ripening since they have different impacts on fruit pigmentation. It will also be interesting to find out if the persistence of chlorophyll affects fruit properties beyond color, in light of a recent study that shows variation in the rice SGR promoter can increase grain yield by delaying crop senescence and increasing photosynthetic competence (Shin et al., 2020), and to investigate whether there is an effect on yield in addition to quality by targeting different alleles of SGR in citrus. The phenomenon of green/orange sector formation in mutant chimeric fruits may also require exploration from both developmental patterning and metabolic regulation aspects. The nature of the inhibition of PSY activity by SGR should be further explored to understand how this interaction affects carotenoid accumulation, and the differences between two truncated versions of SGR, SGRa^STOP, and SGRb, will provide more information about this interaction.