Yafei Qi

Arabidopsis CSP41 proteins form multimeric complexes that bind and stabilize distinct plastid transcripts

The spinach CSP41 protein has been shown to bind and cleave chloroplast RNA in vitro. Arabidopsis thaliana, like other photosynthetic eukaryotes, encodes two copies of this protein. Several functions have been described for CSP41 proteins in Arabidopsis, including roles in chloroplast rRNA metabolism and transcription. CSP41a and CSP41b interact physically, but it is not clear whether they have distinct functions. It is shown here that CSP41b, but not CSP41a, is an essential and major component of a specific subset of RNA-binding complexes that form in the dark and disassemble in the light. RNA immunoprecipitation and hybridization to gene chips (RIP-chip) experiments indicated that CSP41 complexes can contain chloroplast mRNAs coding for photosynthetic proteins and rRNAs (16S and 23S), but no tRNAs or mRNAs for ribosomal proteins. Leaves of plants lacking CSP41b showed decreased steady-state levels of CSP41 target RNAs, as well as decreased plastid transcription and translation rates. Representative target RNAs were less stable when incubated with broken chloroplasts devoid of CSP41 complexes, indicating that CSP41 proteins can stabilize target RNAs. Therefore, it is proposed that (i) CSP41 complexes may serve to stabilize non-translated target mRNAs and precursor rRNAs during the night when the translational machinery is less active in a manner responsive to the redox state of the chloroplast, and (ii) that the defects in translation and transcription in CSP41 protein-less mutants are secondary effects of the decreased transcript stability.

The PHOTOSYNTHESIS AFFECTED MUTANT68–LIKE Protein Evolved from a PSII Assembly Factor to Mediate Assembly of the Chloroplast NAD(P)H Dehydrogenase Complex in Arabidopsis

Assembly factors are auxiliary proteins that promote the biogenesis of multiprotein complexes without being subunits of those. PHOTOSYNTHESIS-AFFECTED MUTANT68-LIKE (PAM68L) operates as an assembly factor of the NAD(P)H dehydrogenase complex in chloroplasts of flowering plants and derives evolutionarily from PAM68, an assembly factor of photosystem II in cyanobacteria, mosses, and plants. In vascular plants, the chloroplast NAD(P)H dehydrogenase complex (NDH-C) is assembled from five distinct subcomplexes, the membrane-spanning (subM) and the luminal (subL) subcomplexes, as well as subA, subB, and subE. The assembly process itself is poorly understood. Vascular plant genomes code for two related intrinsic thylakoid proteins, PHOTOSYNTHESIS-AFFECTED MUTANT68 (PAM68), a photosystem II assembly factor, and PHOTOSYNTHESIS-AFFECTED MUTANT68-LIKE (PAM68L). As we show here, inactivation of Arabidopsis thaliana PAM68L in the pam68l-1 mutant identifies PAM68L as an NDH-C assembly factor. The mutant lacks functional NDH holocomplexes and accumulates three distinct NDH-C assembly intermediates (subB, subM, and subA+L), which are also found in mutants defective in subB assembly (ndf5) or subM expression (CHLORORESPIRATORY REDUCTION4-3 mutant). NDH-C assembly in the cyanobacterium Synechocystis sp PCC 6803 and the moss Physcomitrella patens does not require PAM68 proteins, as demonstrated by the analysis of knockout lines for the single-copy PAM68 genes in these species. We conclude that PAM68L mediates the attachment of subB- and subM-containing intermediates to a complex that contains subA and subL. The evolutionary appearance of subL and PAM68L during the transition from mosses like P. patens to flowering plants suggests that the associated increase in the complexity of the NDH-C might have been facilitated by the recruitment of evolutionarily novel assembly factors like PAM68L.

Mutations in circularly permuted GTPase family genes AtNOA1/RIF1/SVR10 and BPG2 suppress var2-mediated leaf variegation in Arabidopsis thaliana

Leaf variegation mutants constitute a unique group of chloroplast development mutants and are ideal genetic materials to dissect the regulation of chloroplast development. We have utilized the Arabidopsis yellow variegated (var2) mutant and genetic suppressor analysis to probe the mechanisms of chloroplast development. Here we report the isolation of a new var2 suppressor locus SUPPRESSOR OF VARIEGATION (SVR10). Genetic mapping and molecular complementation indicated that SVR10 encodes a circularly permuted GTPase that has been reported as Arabidopsis thaliana NITRIC OXIDE ASSOCIATED 1 (AtNOA1) and RESISTANT TO INHIBITION BY FOSMIDOMYCIN 1 (RIF1). Biochemical evidence showed that SVR10/AtNOA1/RIF1 likely localizes to the chloroplast stroma. We further demonstrate that the mutant of a close homologue of SVR10/AtNOA1/RIF1, BRASSINAZOLE INSENSITIVE PALE GREEN 2 (BPG2), can also suppress var2 leaf variegation. Mutants of SVR10 and BPG2 are impaired in photosynthesis and the accumulation of chloroplast proteins. Interestingly, two-dimensional blue native gel analysis showed that mutants of SVR10 and BPG2 display defects in the assembly of thylakoid membrane complexes including reduced levels of major photosynthetic complexes and the abnormal accumulation of a chlorophyll-protein supercomplex containing photosystem I. Taken together, our findings suggest that SVR10 and BPG2 are functionally related with VAR2, likely through their potential roles in regulating chloroplast protein homeostasis, and both SVR10 and BPG2 are required for efficient thylakoid protein complex assembly and photosynthesis.

Chloroplast translation initiation factors regulate leaf variegation and development

SVR9 and its homolog SVR9L1 encode functionally redundant chloroplast translation initiation factors essential for chloroplast and leaf development in Arabidopsis. Chloroplast development requires the coordinated expressions of nuclear and chloroplast genomes, and both anterograde and retrograde signals exist and work together to facilitate this coordination. We have utilized the Arabidopsis yellow variegated (var2) mutant as a tool to dissect the genetic regulatory network of chloroplast development. Here, we report the isolation of a new (to our knowledge) var2 genetic suppressor locus, SUPPRESSOR OF VARIEGATION9 (SVR9). SVR9 encodes a chloroplast-localized prokaryotic type translation initiation factor 3 (IF3). svr9-1 mutant can be fully rescued by the Escherichia coli IF3 infC, suggesting that SVR9 functions as a bona fide IF3 in the chloroplast. Genetic and molecular evidence indicate that SVR9 and its close homolog SVR9-LIKE1 (SVR9L1) are functionally interchangeable and their combined activities are essential for chloroplast development and plant survival. Interestingly, we found that SVR9 and SVR9L1 are also involved in normal leaf development. Abnormalities in leaf anatomy, cotyledon venation patterns, and leaf margin development were identified in svr9-1 and mutants that are homozygous for svr9-1 and heterozygous for svr9l1-1 (svr9-1 svr9l1-1/+). Meanwhile, as indicated by the auxin response reporter DR5:GUS, auxin homeostasis was disturbed in svr9-1, svr9-1 svr9l1-1/+, and plants treated with inhibitors of chloroplast translation. Genetic analysis established that SVR9/SVR9L1-mediated leaf margin development is dependent on CUP-SHAPED COTYLEDON2 activities and is independent of their roles in chloroplast development. Together, our findings provide direct evidence that chloroplast IF3s are essential for chloroplast development and can also regulate leaf development.

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.

The High Light Response and Redox Control of Thylakoid FtsH Protease in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, the major protease involved in the maintenance of photosynthetic machinery in thylakoid membranes, the FtsH protease, mostly forms large hetero-oligomers (∼1 MDa) comprising FtsH1 and FtsH2 subunits, whatever the light intensity for growth. Upon high light exposure, the FtsH subunits display a shorter half-life, which is counterbalanced by an increase in FTSH1/2 mRNA levels, resulting in the modest upregulation of FtsH1/2 proteins. Furthermore, we found that high light increases the protease activity through a hitherto unnoticed redox-controlled reduction of intermolecular disulfide bridges. We isolated a Chlamydomonas FTSH1 promoter-deficient mutant, ftsh1-3, resulting from the insertion of a TOC1 transposon, in which the high light-induced upregulation of FTSH1 gene expression is largely lost. In ftsh1-3, the abundance of FtsH1 and FtsH2 proteins are loosely coupled (decreased by 70% and 30%, respectively) with no formation of large and stable homo-oligomers. Using strains exhibiting different accumulation levels of the FtsH1 subunit after complementation of ftsh1-3, we demonstrate that high light tolerance is tightly correlated with the abundance of the FtsH protease. Thus, the response of Chlamydomonas to light stress involves higher levels of FtsH1/2 subunits associated into large complexes with increased proteolytic activity.

Mutations in the Arabidopsis AtMRS2-11/AtMGT10/VAR5 gene cause leaf reticulation

In higher plants, the development of functional chloroplasts is essential for photosynthesis and many other physiological processes. With a long-term goal of elucidating the genetic regulation of chloroplast development, we identified two allelic leaf variegation mutants, variegated5-1 (var5-1) and var5-2. Both mutants showed a distinct leaf reticulation phenotype of yellow paraveinal regions and green interveinal regions, and the leaf reticulation phenotype correlated with photosynthetic defects. Through the identification of mutation sites in the two mutant alleles and the molecular complementation, we confirmed that VAR5 encodes a CorA family of Mg2+ transporters also known as AtMRS2-11/AtMGT10. Using protoplast transient expression and biochemical fractionation assays, we demonstrated that AtMRS2-11/AtMGT10/VAR5 likely localizes to the chloroplast envelope. Moreover, we established that AtMRS2-11/AtMGT10/VAR5 forms large molecular weight complexes in the chloroplast and the sizes of these complexes clearly exceed those of their bacterial counterparts, suggesting the compositions of CorA Mg2+ transporter complex is different between the chloroplast and bacteria. Our findings indicate that AtMRS2-11/AtMGT10/VAR5 plays an important role in the tissue specific regulation of chloroplast development.

The Cloning and Functional Characterization of Peach CONSTANS and FLOWERING LOCUS T Homologous Genes PpCO and PpFT

Flowering is an essential stage of plant growth and development. The successful transition to flowering not only ensures the completion of plant life cycles, it also serves as the basis for the production of economically important seeds and fruits. CONSTANS (CO) and FLOWERING LOCUS T (FT) are two genes playing critical roles in flowering time control in Arabidopsis. Through homology-based cloning and rapid-amplifications of cDNA ends (RACE), we obtained full-lengths cDNA sequences of Prunus persica CO (PpCO) and Prunus persica FT (PpFT) from peach (Prunus persica (L.) Batsch) and investigated their functions in flowering time regulation. PpCO and PpFT showed high homologies to Arabidopsis CO and FT at DNA, mRNA and protein levels. We showed that PpCO and PpFT were nucleus-localized and both showed transcriptional activation activities in yeast cells, consistent with their potential roles as transcription activators. Moreover, we established that the over-expression of PpCO could restore the late flowering phenotype of the Arabidopsis co-2 mutant, and the late flowering defect of the Arabidopsis ft-1 mutant can be rescued by the over-expression of PpFT, suggesting functional conservations of CO and FT genes in peach and Arabidopsis. Our results suggest that PpCO and PpFT are homologous genes of CO and FT in peach and they may function in regulating plant flowering time.

The Over-Expression of Two Transcription Factors, ABS5/bHLH30 and ABS7/MYB101, Leads to Upwardly Curly Leaves

Proper leaf development is essential for plant growth and development, and leaf morphogenesis is under the control of intricate networks of genetic and environmental cues. We are interested in dissecting these regulatory circuits genetically and report here the isolation of two Arabidopsis dominant mutants, abnormal shoot5-1D (abs5-1D) and abs7-1D identified through activation tagging screens. Both abs5-1D and abs7-1D display an intriguing upwardly curly leaf phenotype. Molecular cloning showed that the elevated expression of a bHLH transcription factor ABS5/T5L1/bHLH30 or a MYB transcription factor ABS7/MYB101 is the cause for the abnormal leaf phenotypes found in abs5-1D or abs7-1D, respectively. Protoplast transient expression assays confirmed that both ABS5/T5L1 and ABS7/MYB101 are targeted to the nucleus. Interestingly, the expression domains of auxin response reporter DR5::GUS were abnormal in leaves of abs5-1D and ABS5/T5L1 over-expression lines. Moreover, cotyledon venation analysis showed that more areoles and free-ending veins are formed in abs5-1D. We found that the epidermis-specific expressions of ABS5/T5L1 or ABS7/MYB101 driven by the Arabidopsis Meristem Layer 1 promoter (PAtML1) were sufficient to recapitulate the curly leaf phenotype of abs5-1D or abs7-1D. In addition, PAtML1::ABS5 lines exhibited similar changes in DR5::GUS expression patterns as those found in 35S-driven ABS5/T5L1 over-expression lines. Our work demonstrated that enhanced expressions of two transcription factors, ABS5/T5L1 and ABS7/MYB101, are able to alter leaf lamina development and reinforce the notion that leaf epidermis plays critical roles in regulating plant organ morphogenesis.

Balance between Cytosolic and Chloroplast Translation Affects Leaf Variegation

Mutations in cytosolic ribosomal protein RPS21 enhance leaf variegation in Arabidopsis by loss of chloroplast VAR2/AtFtsH2 protein, an effect dependent on chloroplast translation. The development of functional chloroplasts relies on the fine coordination of expressions of both nuclear and chloroplast genomes. We have been using the Arabidopsis (Arabidopsis thaliana) yellow variegated (var2) leaf variegation mutant as a tool to dissect the regulation of chloroplast development. In this work, we screened for var2 genetic enhancer modifiers termed enhancer of variegation (evr) mutants and report the characterization of the first EVR locus, EVR1. We showed that EVR1 encodes the cytosolic 80S ribosome 40S small subunit protein RPS21B and the loss of EVR1 causes the enhancement of var2 leaf variegation. We further demonstrated that combined S21 activities from EVR1 and its close homolog, EVR1L1, are essential for Arabidopsis, and they act redundantly in regulating leaf development and var2 leaf variegation. Moreover, using additional cytosolic ribosomal protein mutants, we showed that although mutations in cytosolic ribosomal proteins all enhance var2 leaf variegation to varying degrees, the 40S subunit appears to have a more profound role over the 60S subunit in regulating VAR2-mediated chloroplast development. Comprehensive genetic analyses with var2 suppressors that are defective in chloroplast translation established that the enhancement of var2 leaf variegation by cytosolic ribosomal protein mutants is dependent on chloroplast translation. Based on our data, we propose a model that incorporates the suppression and enhancement of var2 leaf variegation, and hypothesize that VAR2/AtFtsH2 may be intimately involved in the balancing of cytosolic and chloroplast translation programs during chloroplast biogenesis.