The Arabidopsis Spontaneous Cell Death1 gene, encoding a ζ-carotene desaturase essential for carotenoid biosynthesis, is involved in chloroplast development, photoprotection and retrograde signalling

Carotenoids, a class of natural pigments found in all photosynthetic organisms, are involved in a variety of physiologi-cal processes, including coloration, photoprotection, biosynthesis of abscisic acid (ABA) and chloroplast biogenesis.Although carotenoid biosynthesis has been well studied biochemically, the genetic basis of the pathway is not wellunderstood. Here, we report the characterization of two allelic Arabidopsis mutants, spontaneous cell death 1-1 (spcl-1)and spcl-2. The weak allele spcl-1 mutant showed characteristics of bleached leaves, accumulation of superoxide andmosaic cell death. The strong mutant allele spcl-2 caused a complete arrest of plant growth and development shortlyafter germination, leading to a seedling-lethal phenotype. Genetic and molecular analyses indicated that SPC1 encodesa putative ζ-carotene desaturase (ZDS) in the carotenoid biosynthesis pathway. Analysis of carotenoids revealed thatseveral major carotenoid compounds downstream of SPC1/ZDS were substantially reduced in spcl-1, suggesting thatSPC1 is a functional ZDS. Consistent with the downregulated expression of CAO and PORB, the chlorophyll contentwas decreased in spcl-1 plants. In addition, expression of Lhcb1.1, Lhcb1.4 and RbcS was absent in spcl-2, suggestingthe possible involvement of carotenoids in the plastid-to-nucleus retrograde signaling. The spcl-1 mutant also displaysan ABA-deficient phenotype that can be partially rescued by the externally supplied phytohormone. These results suggestthat SPC1/ZDS is essential for biosynthesis of carotenoids and plays a crucial role in plant growth and development.

Decreased glutathione reductase2 leads to early leaf senescence in Arabidopsis

Glutathione reductase（GR） catalyzes the reduction of glutathione disulfide（GSSG） to reduced glutathione（GSH）and participates in the ascorbate-glutathione cycle, which scavenges H_2O_2. Here, we report that chloroplastic/mitochondrial GR2 is an important regulator of leaf senescence. Seed development of the homozygous gr2 knockout mutant was blocked at the globular stage. Therefore, to investigate the function of GR2 in leaf senescence, we generated transgenic Arabidopsis plants with decreased GR2 using RNAi. The GR2 RNAi plants displayed early onset of age-dependent and darkand H2O2-induced leaf senescence, which was accompanied by the induction of the senescence-related marker genes SAG12 and SAG13. Furthermore, transcriptome analysis revealed that genes related to leaf senescence, oxidative stress, and phytohormone pathways were upregulated directly before senescence in RNAi plants. In addition, H2O2 accumulated to higher levels in RNAi plants than in wild-type plants and the levels of H_2O_2 peaked in RNAi plants directly before the early onset of leaf senescence. RNAi plants showed a greater decrease in GSH/GSSG levels than wild-type plants during leaf development. Our results suggest that GR2 plays an important role in leaf senescence by modulating H_2O_2 and glutathione signaling in Arabidopsis.

Chloroplast gene expression:Recent advances and perspectives

Chloroplasts evolved from an ancient cyanobacterial endosymbiont more than 1.5 billion years ago.During subsequent coevolution with the nuclear genome,the chloroplast genome has remained independent,albeit strongly reduced,with its own transcriptional machinery and distinct features,such as chloroplast-specific innovations in gene expression and complicated post-transcriptional processing.Light activates the expression of chloroplast genes via mechanisms that optimize photosynthesis,minimize photodamage,and prioritize energy investments.Over the past few years,studies have moved from describing phases of chloroplast gene expression to exploring the underlying mechanisms.In this review,we focus on recent advances and emerging principles that govern chloroplast gene expression in land plants.We discuss engineering of pentatricopeptide repeat proteins and its biotechnological effects on chloroplast RNA research;new techniques for characterizing the molecular mechanisms of chloroplast gene expression;and important aspects of chloroplast gene expression for improving crop yield and stress tolerance.We also discuss biological and mechanistic questions that remain to be answered in the future.

Genetic analysis of tolerance to photo-oxidative stress induced by high light in winter wheat (Triticum aestivum L.)

High light induced photooxidation （HLIP） usually leads to leaf premature senescence and causes great yield loss in winter wheat. In order to explore the genetic control of wheat tolerance to HLIP stress, a quantitative trait loci （QTL） analysis was conducted on a set of doubled haploid population, derived from two winter wheat cultivars. Actual values of chlorophyll content （Chl）, minimum fluorescence level （Fo）, maximum fluorescence level （Fm）, and the maximum quan^m efficiency of photosystem II （Fv/Fm） under both HLIP and non-stress conditions as well as the ratios of HLIP to non-stress were evaluated. HLIP considerably reduced Chl, Fm, and Fv/Fm, but in- creased Fo, compared with that under non-stress condition. A total of 27, 16, and 28 QTLs were associated with the investigated traits under HLIP and non-stress and the ratios of HLIP to non-stress, respectively. Most of the QTLs for the ratios of HLIP to non-stress collocated or nearly linked with those detected under HLIP condition. HLIP-induced QTLs were mapped on 15 chromosomes, involving in 1A, 1B, 1D, 2A, 2B, 2D, 3A, 3B, 4A, 4D, 5B, 6A, 6B, 7A, and 7D while those expressed under non-stress condition involved in nine chromosomes, includ- ing 1B, 1D, 2A, 2B, 3B, 4A, 5A, 5B, and 7A. The expression patterns of QTLs under HLIP condition were different from that under non-stress condition except for six loci on five chromosomes. The phenotypic variance explained by individual QTL ranged from 5.0% to 19.7% under HLIP, 8.3% to 20.8% under non-stress, and 4.9% to 20.2% for the ratios of HLIP to non-stress, respectively. Some markers, for example, Xgwm192 and WMC331 on 4D regulating Chl, Fo, Fm, and Fv/Fm under HLIP condition, might be used in marker assistant selection.

mTERF5 Acts as a Transcriptional Pausing Factor to Positively Regulate Transcription of Chloroplast psbEFLJ

RNA polymerase transcriptional pausing represents a major checkpoint in transcription in bacteria and metazoans,but it is unknown whether this phenomenon occurs in plant organelles.Here,we report that transcriptional pausing occurs in chloroplasts.We found that mTERF5 specifically and positively regulates the transcription of chloroplast psbEFLJ in Arabidopsis thaliana that encodes four key subunits of photosystem II.We found that mTERF5 causes the plastid-encoded RNA polymerase(PEP)complex to pause at psbEFLJ by binding to the+30 to+51 region of double-stranded DNA.Moreover,we revealed that mTERF5 interacts with pTAC6,an essential subunit of the PEP complex,although pTAC6 is not involved in the transcriptional pausing at psbEFLJ.We showed that mTERF5 recruits additional pTAC6 to the transcriptionally paused region of psbEFLJ,and the recruited pTAC6 proteins could be assembled into the PEP complex to regulate psbEFLJ transcription.Taken together,our findings shed light on the role of transcriptional pausing in chloroplast transcription in plants.

The Phytol Phosphorylation Pathway Is Essential for the Biosynthesis of Phylloquinone, which Is Required for Photosystem I Stability in Arabidopsis

Phytyl-diphosphate, which provides phytyl moieties as a common substrate in both tocopherol and phyllo- quinone biosynthesis, derives from de novo isoprenoid biosynthesis or a salvage pathway via phytol phos- phorylation. However, very little is known about the role and origin of the phytyl moiety for phylloquinone biosynthesis. Since VTE6, a phytyl-phosphate kinase, is a key enzyme for phytol phosphorylation, we char- acterized Arabidopsis vte6 mutants to gain insight into the roles of phytyl moieties in phylloquinone biosyn- thesis and of phylloquinone in photosystem I （PSI） biogenesis. The VTE6 knockout mutants vte6-1 and vte6-2 lacked detectable phylloquinone, whereas the phylloquinone content in the VTE6 knockdown mutant vte6-3 was 90% lower than that in wild-type. In vte6 mutants, PSI function was impaired and accu- mulation of the PSI complex was defective. The PSI core subunits PsaA/B were efficiently synthesized and assembled into the PSI complex in vte6-3. However, the degradation rate of PSI subunits in the assembled PSI complex was more rapid in vte6-3 than in wild-type. In vte6-3, PSI was more susceptible to high-light damage than in wild-type. Our results provide the first genetic evidence that the phytol phosphorylation pathway is essential for phylloquinone biosynthesis, and that phylloquinone is required for PSI complex stability.

Autophagy targets Hd1 for vacuolar degradation to regulate rice flowering

Flowering time(heading date)is a critical agronomic trait that determines the yield and regional adaptability of crops.Heading date 1(Hd1)is a central regulator of photoperiodic flowering in rice(Oryza sativa).However,how the homeostasis of Hd1 protein is achieved is poorly understood.Here,we report that the nuclear autophagy pathway mediates Hd1 degradation in the dark to regulate flowering.Loss of autophagy function results in an accumulation of Hd1 and delays flowering under both short-day and long-day conditions.In the dark,nucleus-localized Hd1 is recognized as a substrate for autophagy and is subjected to vacuolar degradation via the autophagy protein OsATG8.The Hd1-0sATG8 interaction is required for autophagic degradation of Hd1 in the dark.Our study reveals a new mechanism by which Hd1 protein homeostasis is regulated by autophagy to control rice flowering.Our study also indicates that the regulation of flowering by autophagic degradation of Hd1 orthologs may have arisen over the course of mesangiosperm evolution,which would have increased their flexibility and adaptability to the environment by modulating flowering time.

Pentatricopeptide repeat protein PHOTOSYSTEM I BIOGENESIS FACTOR2 is required for splicing of ycf3

To gain a better understanding of the molec-ular mechanisms of photosystem!(PSI)biogenesis,wecharacterized the Arabidopsis thaliana photosystem l bio-genesis factor 2(pbf2)mutant,which lacks PSl complex.PBF2 encodes a P-class pentatricopeptide repeat(PPR)protein.In the pbf2 mutants,we observed a striking de-crease in the transcript level of only one gene,thechloroplast gene ycf3,which is essential for PSl assembly.Further analysis of ycf3 transcripts showed that PBF2 isspecifically required for the splicing of ycf3 intron 1.Computational prediction of binding sequences and elec-trophoretic mobility shift assays reveal that PBF2 specifi-cally binds to a sequence in ycf3 intron 1.Moreover,we found that PBF2 interacted with two general factors forgroup ll intron splicing CHLOROPLAST RNA SPLICING2-AsSOCIATED FACTOR1(CAF1)and CAF2,and facilitated theassociation of these two factors with ycf3 intron 1.Ourresults suggest that PBF2 is specifically required for thesplicing of ycf3 intron 1 through cooperating with CAF1and CAF2.Our results also suggest that additional proteinsare required to contribute to the specificity of CAF-dependent group ll intron splicing.

A Kinase-Phosphatase-Transcription Factor Module Regulates Adventitious Root Emergence in Arabidopsis Root-Hypocotyl Junctions

Adventitious roots form from non-root tissues as part of normal development or in response to stress or wounding.The root primordia form in the source tissue,and during emergence the adventitious roots penetrate the inner cell layers and the epidermis;however,the mechanisms underlying this emergence remain largely unexplored.Here,we report that a regulatory module composed of the AP2/ERF transcription factor ABSCISIC ACID INSENSITIVE 4(ABI4),the MAP kinases MPK3 and MPK6,and the phosphatase PP2C12 plays an important role in the emergence of junction adventitious roots(J-ARs)from the root-hypocotyl junctions in Arabidopsis thaliana.ABI4 negatively regulates J-AR emergence,preventing the accumulation of reactive oxygen species and death of epidermal cells,which would otherwise facilitate J-AR emergence.Phosphorylation by MPK3/MPK6 activates ABI4 and dephosphorylation by PP2C12 inactivates ABI4.MPK3/MPK6 also directly phosphorylate and inactivate PP2C12 during J-AR emergence.We propose that this"double-check"mechanism increases the robustness of MAP kinase signaling and finely regulates the local programmed cell death required for J-AR emergence.