During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll → pheophytin → pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.

During senescence, chlorophyll (chl) is metabolized to colorless nonfluorescent chl catabolites (NCCs). A central reaction of the breakdown pathway is the ring cleavage of pheophorbide (pheide) a to a primary fluorescent chl catabolite. Two enzymes catalyze this reaction, pheide a oxygenase (PAO) and red chl catabolite reductase. Five NCCs and three fluorescent chl catabolites (FCCs) accumulated during dark-induced chl breakdown in Arabidopsis (Arabidopsis thaliana). Three of these NCCs and one FCC (primary fluorescent chl catabolite-1) were identical to known catabolites from canola (Brassica napus). The presence in Arabidopsis of two modified FCCs supports the hypothesis that modifications, as present in NCCs, occur at the level of FCC. Chl degradation in Arabidopsis correlated with the accumulation of FCCs and NCCs, as well as with an increase in PAO activity. This increase was due to an up-regulation of Pao gene expression. In contrast, red chl catabolite reductase is not regulated during leaf development and senescence. A pao1 knockout mutant was identified and analyzed. The mutant showed an age- and light-dependent cell death phenotype on leaves and in flowers caused by the accumulation of photoreactive pheide a. In the dark, pao1 exhibited a stay-green phenotype. The key role of PAO in chl breakdown is discussed.

Our most productive crops and native vegetation use a modified version of photosynthesis known as the C₄ pathway. Leaves of C₄ crops have increased nitrogen and water use efficiencies compared with C₃ species. Although the modifications to leaves of C₄ plants are complex, their faster growth led to the proposal that C₄ photosynthesis should be installed in C₃ crops in order to increase yield potential. Typically, a limited set of proteins become restricted to mesophyll or bundle sheath cells, and this allows CO₂ to be concentrated around the primary carboxylase RuBisCO. The role that these proteins play in C₃ species prior to their recruitment into the C₄ pathway is addressed here. Understanding the role of these proteins in C₃ plants is likely to be of use in predicting how the metabolism of a C₃ leaf will alter as components of the C₄ pathway are introduced as part of efforts to install characteristics of C₄ photosynthesis in leaves of C₃ crops.

A central reaction of chlorophyll breakdown, porphyrin ring opening of pheophorbide a to the primary fluorescent chlorophyll catabolite (pFCC), requires pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR), with red chlorophyll catabolite (RCC) as a presumably PAO-bound intermediate. In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). Here, we show that RCCR-deficient Arabidopsis thaliana accumulates RCC and three RCC-like pigments during senescence, as well as FCCs and NCCs. We also show that the stereospecificity of Arabidopsis RCCR is defined by a small protein domain and can be reversed by a single Phe-to-Val exchange. Exploiting this feature, we prove the in vivo participation of RCCR in chlorophyll breakdown. After complementation of RCCR mutants with RCCRs exhibiting alternative specificities, patterns of chlorophyll catabolites followed the specificity of complementing RCCRs. Light-dependent leaf cell death observed in different RCCR-deficient lines strictly correlated with the accumulation of RCCs and the release of singlet oxygen, and PAO induction preceded lesion formation. These findings suggest that RCCR absence causes leaf cell death as a result of the accumulation of photodynamic RCC. We conclude that RCCR (together with PAO) is required for the detoxification of chlorophyll catabolites and discuss the biochemical role(s) for this enzyme.

A key gene involved in plant senescence, mutations of which partially disable chlorophyll catabolism and confer stay-green leaf and cotyledon phenotypes, has been identified in Pisum sativum , Arabidopsis thaliana , and Festuca pratensis by using classical and molecular genetics and comparative genomics. A stay-green locus in F. pratensis is syntenically equivalent to a similar stay-green locus on rice chromosome 9. Functional testing in Arabidopsis of a homolog of the rice candidate gene revealed (i) senescence-associated gene expression and (ii) a stay-green phenotype after RNA interference silencing. Genetic mapping in pea demonstrated cosegregation with the yellow/green cotyledon polymorphism ( I/i ) first reported by Gregor Mendel in 1866.

With at least 60 independent origins spanning monocotyledons and dicotyledons, the C₄ photosynthetic pathway represents one of the most remarkable examples of convergent evolution. The recurrent evolution of this highly complex trait involving alterations to leaf anatomy, cell biology and biochemistry allows an increase in productivity by ∼50% in tropical and subtropical areas. The extent to which separate lineages of C₄ plants use the same genetic networks to maintain C₄ photosynthesis is unknown. We developed a new informatics framework to enable deep evolutionary comparison of gene expression in species lacking reference genomes. We exploited this to compare gene expression in species representing two independent C₄ lineages (Cleome gynandra and Zea mays) whose last common ancestor diverged ∼140 million years ago. We define a cohort of 3,335 genes that represent conserved components of leaf and photosynthetic development in these species. Furthermore, we show that genes encoding proteins of the C₄ cycle are recruited into networks defined by photosynthesis-related genes. Despite the wide evolutionary separation and independent origins of the C₄ phenotype, we report that these species use homologous transcription factors to both induce C₄ photosynthesis and to maintain the cell specific gene expression required for the pathway to operate. We define a core molecular signature associated with leaf and photosynthetic maturation that is likely shared by angiosperm species derived from the last common ancestor of the monocotyledons and dicotyledons. We show that deep evolutionary comparisons of gene expression can reveal novel insight into the molecular convergence of highly complex phenotypes and that parallel evolution of trans-factors underpins the repeated appearance of C₄ photosynthesis. Thus, exploitation of extant natural variation associated with complex traits can be used to identify regulators. Moreover, the transcription factors that are shared by independent C₄ lineages are key targets for engineering the C₄ pathway into C₃ crops such as rice. C₄ photosynthesis is one of the most successful and widespread examples of convergent evolution; the first C₄ plant evolved long after the extinction of the dinosaurs, yet C₄ species now account for ∼30% of primary productivity on earth. Compared with ancestral C₃ photosynthesis, the C₄ pathway allows faster rates of growth, and thus international efforts have been mustered to introduce advantageous C₄ traits into important C₃ crops to increase their yield. However, the transition from C₃ to C₄ involves complex alterations to leaf anatomy and biochemistry. Despite these multiple changes, C₄ photosynthesis has evolved independently at least 60 times. Through DNA and RNA sequencing we are beginning define a catalog of genes associated with C₃ or C₄ photosynthesis. However, we know little about how these genes act co-ordinately to bring about the convergent C₄ phenotype. In this work we develop a new informatics framework to reveal that two independent lineages of C₄ plants have co-opted the same regulators of gene expression to generate the C₄ leaf. Our findings provide a new paradigm for investigating the genetics of convergent traits and the origin of convergent phenotypes. Moreover, they reveal significant new insight into the regulatory mechanisms governing the origins of C₄ photosynthesis.