Construction of a chloroplast protein interaction network and functional mining of photosynthetic proteins in Arabidopsis thaliana

Chloroplast is a typical plant cell organelle where photosynthesis takes place. In this study, a total of 1 808 chloroplast core proteins in Arabidopsis thaliana were reliably identified by combining the results of previously published studies and our own predictions. We then constructed a chloroplast protein interaction network primarily based on these core protein interactions. The network had 22 925 protein interaction pairs which involved 2 214 proteins. A total of 160 previously uncharacterized proteins were annotated in this network. The subunits of the photosynthetic complexes were modularized, and the functional relationships among photosystem Ⅰ （PSI）, photosystem Ⅱ （PSII）, light harvesting complex of photosystem Ⅰ （LHC Ⅰ） and light harvesting complex of photosystem Ⅰ （LHC Ⅱ） could be deduced from the predicted protein interactions in this network. We further confirmed an interaction between an unknown protein AT1G52220 and a photosynthetic subunit PSI-D2 by yeast two-hybrid analysis. Our chloroplast protein interaction network should be useful for functional mining of photosynthetic proteins and investigation of chloroplast-related functions at the systems biology level in Arabidopsis.

The contributions of sporophytic tapetum to pollen formation

Successful pollen formation is essential for plant reproduction.During anther development,microspore mother cells undergo meiosis to form tetrads.After being released from the tetrad,microspores develop into mature pollen.The tapetum is the innermost layer of anther somatic cells and forms a locule to provide nutrition,enzymes and pollen wall materials for microspore development.Based on the male sterile phenotype,many genes important for tapetum and pollen development have been cloned.In this review,we highlight the genetic pathway of DYT1-TDF1-AMS-MS188-MS1 which acts in tapetal development in Arabidopsis.We also compared this genetic pathway in different species such as Arabidopsis,rice and maize.Based on this pathway,we review recent findings and insights into the contribution of the tapetum to pollen formation at the molecular level.1)Tapetum provides nutrition for microspore development.2)Tapetum provides enzymes to dissolve pectin and callose to release microspores from tetrads.3)Tapetum synthesizes precursors for pollen wall formation via different molecular pathways.4)Tapetum provides precursors for pollen coat formation.5)Tapetum provides small RNAs to regulate genic methylation in the germline cells.

The regulatory mechanism of rapid lignification for timely anther dehiscence

Anther dehiscence is a crucial event in plant reproduction,tightly regulated and dependent on the lignification of the anther endothecium.In this study,we investigated the rapid lignification process that ensures timely anther dehiscence in Arabidopsis.Our findings reveal that endothecium lignification can be divided into two distinct phases.During Phase Ⅰ,lignin precursors are synthesized without polymerization,while Phase Ⅱ involves simultaneous synthesis of lignin precursors and polymerization.The transcription factors MYB26,NST1/2,and ARF17 specifically regulate the pathway responsible for the synthesis and polymerization of lignin monomers in Phase Ⅱ.MYB26-NST1/2 is the key regulatory pathway responsible for endothecium lignification,while ARF17 facilitates this process by interacting with MYB26.Interestingly,our results demonstrate that the lignification of the endothecium,which occurs within approximately 26 h,is much faster than that of the vascular tissue.These findings provide valuable insights into the regulation mechanism of rapid lignification in the endothecium,which enables timely anther dehiscence and successful pollen release during plant reproduction.

Glycerol-3-Phosphate Acyltransferase 6 （GPAT6） Is Important for Tapetum Development in Arabidopsis and Plays Multiple Roles in Plant Fertility

Glycerol-3-phosphate acyltransferase （GPAT） mediates the initial synthetic step for the formation of glycer- olipids, which act as the major components of biological membranes and the principal stored forms of energy. GPAT6 is a member of the Arabidopsis GPAT family, which is crucial for cutin biosynthesis in sepals and petals. In this work, a func- tional analysis of GPAT6 in anther development and plant fertility was performed. GPAT6 was highly expressed in the tapetum and microspores during anther development. The knockout mutant, gpat6, caused a massive reduction in seed production. This report shows that the ablation of GPAT6 caused defective tapetum development with reduced endoplas- mic reticulum （ER） profiles in the tapetum, which largely led to the abortion of pollen grains and defective pollen wall formation. In addition, pollen germination and pollen tube elongation were affected in the mutant plants. Furthermore, the double mutant analysis showed that GPAT6 and GPAT1 make joint effects on the release of microspores from tetrads and stamen filament elongation. This work shows that GPAT6 plays multiple roles in stamen development and fertility in Arabidopsis.

Arabidopsis AT-hook Protein TEK Positively Regulates the Expression of Arabinogalactan Proteins for Nexine Formation

Nexine is a conserved layer of the pollen wall. We previously reported that the nexine layer is absent in the knockout mutant of Arabidopsis TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK （TEK） gene. In this study, we investigated the molecular regulatory functions of TEK in pollen development and identified the genes encoding Arabinogalactan proteins （AGPs） as direct targets of TEK, which are essential for nexine formation. Phenotypic similarity between tek and the TEK-SRDX transgenic lines suggest that TEK plays a role in transcriptional activation in anther development. Microarray analysis identified a total of 661 genes downregulated in tek, including four genes encoding AGPs, AGP6, AGP11, AGP23, and AGP40. Electrophoretic mobility shift assays showed that TEK could directly bind the nuclear matrix attachment region （MAR） and the promoter of AGP6. Chromatin immunoprecipitation followed by PCR analysis demonstrated that TEK is enriched in the promoters of the four AGP genes. Expression of AGP6 driven by the TEK promoter in tek partially rescued both nexine formation and plant fertility. These results indicate that TEK directly reg- ulates AGP expression in the anther to control nexine layer formation. We also proposed that glycoproteins might be essential components of the nexine laver in the oollen wall.

Pollen wall pattern in Arabidopsis

The pollen wall is a solid and variously sculptured structure. This pattern is determined inside a tetrad. During meiosis, the callose wall is formed outside of the meiocyte/microspore to form a tetrad. Then, primexine is deposited between the callose wall and the microspore plasma membrane which will become undulated. The sporopollenin deposits on top of the undulated membrane and develops into the pollen wall pattern, while the callose wall is gradually degraded. In recent years, much progress has been made in the study of pollen wall pattern formation, at both molecular and genetic levels. In this review,we summarize these achievements mainly in Arabidopsis.

AtECB1/MRL7, a Thioredoxin-Like Fold Protein with Disulfide Reductase Activity, Regulates Chloroplast Gene Expression and Chloroplast Biogenesis in Arabidopsis thaliana

Plastid-encoded RNA polymerase （PEP） is closely associated with numerous factors to form PEP complex for plastid gene expression and chloroplast development. However, it is not clear how PEP complex are regulated in chloroplast. Here, one thioredoxin-like fold protein, Arabidopsis early chloroplast biogenesis 1 （AtECB1）, an allele of MRL7, was identified to regulate PEP function and chloroplast biogenesis. The knockout lines for AtECB1 displayed albino phenotype and impaired chloroplast development. The transcripts of PEP-dependent plastid genes were barely detected, suggesting that the PEP activity is almost lost in atecbl-1. Although AtECB1 was not identified in PEP complex, a yeast two-hybrid assay and pull-down experiments demonstrated that it can interact with Trx Z and FSD3, two intrinsic subunits of PEP complex, respectively. This indicates that AtECB1 may play a regulatory role for PEP-dependent plastid gene expression through these two subunits. AtECB1 contains a βαβαββα structure in the thioredoxin-like fold domain and lacks the typical C-X-X-C active site motif. Insulin assay demonstrated that AtECB1 harbors disulfide reductase activity in vitro using the purified recombinant AtECB1 protein. This showed that this thioredoxin-like fold protein, AtECB1 also has the thioredoxin activity. AtECB1 may play a role in thioredoxin signaling to regulate plastid gene expression and chloroplast development.

Phenylpropanoid Derivatives Are Essential Components of Sporopollenin in Vascular Plants

The outer wall of pollen and spores,namely the exine,is composed of sporopollenin,which is highly resistant to chemical reagents and enzymes.In this study,we demonstrated that phenylpropanoid pathway derivatives are essential components of sporopollenin in seed plants.Spectral analyses showed that the autofluorescence of Lilium and Arabidopsis sporopollenin is similar to that of lignin.Thioacidolysis and NMR analyses of pollen from Lilium and Cryptomeria further revealed that the sporopollenin of seed plants contains phenylpropanoid derivatives,including p-hydroxybenzoate(p-BA),p-coumarate(p-CA),ferulate(FA),and lignin guaiacyl(G)units.The phenylpropanoid pathway is expressed in the tapetum in Arabidopsis,consistent with the fact that the sporopollenin precursor originates from the tapetum.Further germination and comet assays showed that this pathway plays an important role in protection of pollen against UV radiation.In the pteridophyte plant species Ophioglossum vulgatum and Lycopodium clavata,phenylpropanoid derivatives including p-BA and p-CA were also detected,but G units were not.Taken together,our results indicate that phenylpropanoid derivatives are essential for sporopollenin synthesis in vascular plants.In addition,sporopollenin autofluorescence spectra of bryophytes,such as Physcomitrella and Haplocladium,exhibit distinct characteristics compared with those of vascular plants,indicating the diversity of sporopollenin among land plants.

Anther Endothecium-Derived Very-Long-Chain Fatty Acids Facilitate Pollen Hydration in Arabidopsis

Dear Editor,Pollen hydration is a prerequisite for pollen germination and sub- sequent pollen tube growth. As a result of two decades of biochemical studies and genetic identification of the eceriferum （cer） male-sterile mutants, the very-long-chain fatty acid （VLCFA） lipids in the pollen coat have been well known to play a critical role in pollen hydration （Preuss et al., 1993; Fiebig et al., 2000; Hiscock and Allen, 2008）.

A cellular mechanism underlying the restoration of thermo/photoperiod-sensitive genic male sterility

During anther development,the transformation of the microspore into mature pollen occurs under the pro-tection of first the tetrad wall and later the pollen wall.Mutations in genes involved in this wall transition often lead to microspore rupture and male sterility;some such mutants,such as the reversible male sterile(rvms)mutant,are thermo/photoperiod-sensitive genic male sterile(P/TGMS)lines.Previous studies have shown that slow development is a general mechanism of P/TGMS fertility restoration.In this study,we iden-tified restorer of rvms-2(res2),which is an allele of QUARTET3(QRT3)encoding a polygalacturonase that shows delayed degradation of the tetrad pectin wall.We found that MS188,a tapetum-specific transcrip-tion factor essential for pollen wall formation,can activate QRT3 expression for pectin wall degradation,indicating a non-cell-autonomous pathway involved in the regulation of the cell wall transition.Further as-says showed that a delay in degradation of the tetrad pectin wall is responsible for the fertility restoration of rvms and other P/TGMS lines,whereas early expression of QRT3 eliminates low temperature restoration of rvms-2 fertility.Taken together,these results suggest a likely cellular mechanism of fertility restoration in P/TGMS lines,that is,slow development during the cell wall transition of P/TGMS microspores may reduce the requirement for their wall protection and thus support their development into functional pollens,leading to restored fertility.

Regulation of sporopollenin synthesis for pollen wall formation in plant

Pollen wall is the most complicated cell wall in plant. It is composed of outer exine and inner intine with the exine fiar- ther divided into sexine and nexine. The composition of in- tine layer is generally considered to be same as a plant cell wall which is mainly composed of cellulose. Nexine layer can only be observed under transmission electron microscope. Recent investigation suggested that the major composition of nexine is arabinogalactan proteins （Jia et al., 2015）.

Delayed callose degradation restores the fertility of multiple P/TGMS lines in Arabidopsis

Photoperiod/temperature-sensitive genic male sterility(P/TGMS)is widely applied for improving crop production.Previous investigations using the reversible male sterile(rvms)mutant showed that slow development is a general mechanism for restoring fertility to P/TGMS lines in Arabidopsis.In this work,we isolated a restorer of rvms–2(res3),as the male sterility of rvms–2 was rescued by res3.Phenotype analysis and molecular cloning show that a point mutation in UPEX1 l in res3 leads to delayed secretion of callase A6 from the tapetum to the locule and tetrad callose wall degradation.Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis demonstrated that the tapetal transcription factor ABORTED MICROSPORES directly regulates UPEX1 expression,revealing a pathway for tapetum secretory function.Early degradation of the callose wall in the transgenic line eliminated the fertility restoration effect of res3.The fertility of multiple known P/TGMS lines with pollen wall defects was also restored by res3.We propose that the remnant callose wall may broadly compensate for the pollen wall defects of P/TGMS lines by providing protection for pollen formation.A cellular mechanism is proposed to explain how slow development restores the fertility of P/TGMS lines in Arabidopsis.

A Point Mutation in the Pentatricopeptide repeat Motif of the AtECB2 Protein Causes Delayed Chloroplast Development

AtECB2 encodes a pentatricopeptide repeat（PPR） protein that regulates the editing of the plastid genes accD and ndhF.The ecb2-1 knockout shows an albino phenotype and is seedling lethal.In this study, we isolated an allelic mutant of the AtECB2 gene,ecb2-2,which showed delayed greening phenotype but could complete their life cycle.In this mutant,the Thr^500 is converted to Ile^500 in the 13^th PPR motif of the AtECB2 protein.Transmission electron microscopy demonstrated that chloroplast development was delayed in both the cotyledons and leaves of the mutant.An investigation of the chloroplast gene expression profile indicated that PEP（plastid-encoded RNA polymerase） activity in ecb2-2 cotyledons was not obviously affected,whereas it was severely impaired in ecb2-1.This result suggests that the PEP activities cause the different phenotypes of the ecb2-1 and ecb2-2 mutants.The editing efficiency of the three editing sites of accD（C794 and C1568） and ndhF（C290） in the mutant was dynamically altered, which was in agreement with the phenotype.This result indicates that the editing efficiency of accD and ndhF in the ecb2-2 mutant is associated with a delayed greening phenotype.As ecb2-2 can survive and set seeds,this mutant can be used for further investigation of RNA editing and chloroplast development in arabidopsis.