Loss of green color in leaves results from chlorophyll (Chl) degradation in chloroplasts, but little is known about how Chl catabolism is regulated throughout leaf development. Using the staygreen (sgr) mutant in rice (Oryza sativa), which maintains greenness during leaf senescence, we identified Sgr, a senescence-associated gene encoding a novel chloroplast protein. Transgenic rice overexpressing Sgr produces yellowish-brown leaves, and Arabidopsis thaliana pheophorbide a oxygenase-impaired mutants exhibiting a stay-green phenotype during dark-induced senescence have reduced expression of Sgr homologs, indicating that Sgr regulates Chl degradation at the transcriptional level. We show that the leaf staygreenness of the sgr mutant is associated with a failure in the destabilization of the light-harvesting chlorophyll binding protein (LHCP) complexes of the thylakoid membranes, which is a prerequisite event for the degradation of Chls and LHCPs during senescence. Transient overexpression of Sgr in Nicotiana benthamiana and an in vivo pull-down assay show that Sgr interacts with LHCPII, indicating that the Sgr-LHCPII complexes are formed in the thylakoid membranes. Thus, we propose that in senescing leaves, Sgr regulates Chl degradation by inducing LHCPII disassembly through direct interaction, leading to the degradation of Chls and Chl-free LHCPII by catabolic enzymes and proteases, respectively.

SummaryIn order to understand the molecular genetic mechanisms of rice (Oryza sativa) organ development, we studied the narrow leaf2 narrow leaf3 (nal2 nal3; hereafter nal2/3) double mutant, which produces narrow-curly leaves, more tillers, fewer lateral roots, opened spikelets and narrow-thin grains.We found that narrow-curly leaves resulted mainly from reduced lateral-axis outgrowth with fewer longitudinal veins and more, larger bulliform cells. Opened spikelets, possibly caused by marginal deformity in the lemma, gave rise to narrow-thin grains.Map-based cloning revealed that NAL2 and NAL3 are paralogs that encode an identical OsWOX3A (OsNS) transcriptional activator, homologous to NARROW SHEATH1 (NS1) and NS2 in maize and PRESSED FLOWER in Arabidopsis. OsWOX3A is expressed in the vascular tissues of various organs, where nal2/3 mutant phenotypes were displayed. Expression levels of several leaf development-associated genes were altered in nal2/3, and auxin transportrelated genes were significantly changed, leading to pin mutant-like phenotypes such as more tillers and fewer lateral roots.OsWOX3A is involved in organ development in rice, lateral-axis outgrowth and vascular patterning in leaves, lemma and palea morphogenesis in spikelets, and development of tillers and lateral roots.

Photosynthetic organisms exhibit a green color due to the accumulation of chlorophyll pigments in chloroplasts. Mg-protoporphyrin IX chelatase (Mg-chelatase) comprises three subunits (ChlH, ChlD and ChlI) and catalyzes the insertion of Mg(2+) into protoporphyrin IX, the last common intermediate precursor in both chlorophyll and heme biosyntheses, to produce Mg-protoporphyrin IX (MgProto). Chlorophyll deficiency in higher plants results in chlorina (yellowish-green) phenotype. To date, 10 chlorina (chl) mutants have been isolated in rice, but the corresponding genes have not yet been identified. Rice Chl1 and Chl9 genes were mapped to chromosome 3 and isolated by map-based cloning. A missense mutation occurred in a highly conserved amino acid of ChlD in the chl1 mutant and ChlI in the chl9 mutant. Ultrastructural analyses have revealed that the grana are poorly stacked, resulting in the underdevelopment of chloroplasts. In the seedlings fed with aminolevulinate-dipyridyl in darkness, MgProto levels in the chl1 and chl9 mutants decreased up to 25% and 31% of that in wild-type, respectively, indicating that the Mg-chelatase activity is significantly reduced, causing the eventual decrease in chlorophyll synthesis. Furthermore, Northern blot analysis indicated that the nuclear genes encoding the three subunits of Mg-chelatase and LhcpII in chl1 mutant are expressed about 2-fold higher than those in WT, but are not altered in the chl9 mutant. This result indicates that the ChlD subunit participates in negative feedback regulation of plastid-to-nucleus in the expression of nuclear genes encoding chloroplast proteins, but not the ChlI subunit.

These authors contributed equally to the work. SUMMARYNADPH:protochlorophyllide oxidoreductase (POR) catalyzes photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in chlorophyll (Chl) synthesis, and is required for prolamellar body (PLB) formation in etioplasts. Rice faded green leaf (fgl) mutants develop yellow/white leaf variegation and necrotic lesions during leaf elongation in field-grown plants. Map-based cloning revealed that FGL encodes OsPORB, one of two rice POR isoforms. In fgl, etiolated seedlings contained smaller PLBs in etioplasts, and lower levels of total and photoactive Pchlide. Under constant or high light (HL) conditions, newly emerging green leaves rapidly turned yellow and formed lesions. Increased levels of non-photoactive Pchlide, which acts as a photosensitizer, may cause reactive oxygen accumulation and lesion formation. OsPORA expression is repressed by light and OsPORB expression is regulated in a circadian rhythm in short-day conditions. OsPORA was expressed at high levels in developing leaves and decreased dramatically in fully mature leaves, whereas OsPORB expression was relatively constant throughout leaf development, similar to expression patterns of AtPORA and AtPORB in Arabidopsis. However, OsPORB expression is rapidly upregulated by HL treatment, similar to the fluence rate-dependent regulation of AtPORC. This suggests that OsPORB function is equivalent to both AtPORB and AtPORC functions. Our results demonstrate that OsPORB is essential for maintaining light-dependent Chl synthesis throughout leaf development, especially under HL conditions, whereas OsPORA mainly functions in the early stages of leaf development. Developmentally and physiologically distinct roles of monocot OsPORs are discussed by comparing with those of dicot AtPORs.

Chlorophyll (Chl) degradation causes leaf yellowing during senescence or under stress conditions. For Chl breakdown, STAY-GREEN1 (SGR1) interacts with Chl catabolic enzymes (CCEs) and light-harvesting complex II (LHCII) at the thylakoid membrane, possibly to allow metabolic channeling of potentially phototoxic Chl breakdown intermediates. Among these Chl catabolic components, SGR1 acts as a key regulator of leaf yellowing. In addition to SGR1 (At4g22920), the Arabidopsis thaliana genome contains an additional homolog, SGR2 (At4g11910), whose biological function remains elusive. Under senescence-inducing conditions, SGR2 expression is highly up-regulated, similarly to SGR1 expression. Here we show that SGR2 function counteracts SGR1 activity in leaf Chl degradation; SGR2-overexpressing plants stayed green and the sgr2-1 knockout mutant exhibited early leaf yellowing under age-, dark-, and stress-induced senescence conditions. Like SGR1, SGR2 interacted with LHCII but, in contrast to SGR1, SGR2 interactions with CCEs were very limited. Furthermore, SGR1 and SGR2 formed homo- or heterodimers, strongly suggesting a role for SGR2 in negatively regulating Chl degradation by possibly interfering with the proposed CCE-recruiting function of SGR1. Our data indicate an antagonistic evolution of the functions of SGR1 and SGR2 in Arabidopsis to balance Chl catabolism in chloroplasts with the dismantling and remobilizing of other cellular components in senescing leaf cells.