Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis

Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene （PDS3） encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarfphenotypes ofthepds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in thepds3 mutant. Further analysis showed that high level ofphytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype ofthepds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.

Generation of a series of mutant lines resistant to imidazolinone by screening an EMS-based mutant library in common wheat

The breeding of herbicide-resistant wheat varieties has helped control weeds in wheat fields economically and effectively.Imidazolinone (IMI) herbicides are popular as they have low toxicity in mammals,are effective at small doses,and exhibit broad-spectrum herbicidal action in the field.Therefore,the isolation and genetic and molecular characterization of IMI-resistant wheat mutants will enhance weed management in wheat fields.In the present study,352 IMI-resistant plants were isolated by genetic screening from a mutant pool prepared by EMS-based random mutagenesis.Cloning of the mutated genes from the IMI-resistant plants indicated that ten taals alleles had been isolated,and mutation in one of three Ta ALS homolog genes conferred IMI resistance,and such a mutation is a dominant trait.Further analysis showed that taals-d exhibited the greatest IMI resistance,whereas taals-b exhibited the weakest resistance to IMI among three homologous taals mutants.In terms of IMI resistance,the taals triple mutant was stronger than the taals double mutants,and the taals double mutants were stronger than the single mutants,indicating a dose-dependent effect of the Ta ALS mutation on IMI resistance in wheat.Biochemical analysis indicated that the mutation in Ta ALS increased the tolerance of Ta ALS to inhibition by IMI.Our work details the genetic and molecular characterization of als wheat mutants,provides a foundation for understanding IMI resistance and breeding wheat varieties with herbicide resistance,and indicates that genetic screening using a mutagenized pool is an effective and important means of breeding crops with additional desired agricultural traits.

Current strategies and advances in wheat biology

The characterization of agronomically important genes has great potential for the improvement of wheat.However,progress in wheat genetics and functional genomics has been impeded by the high complexity and enormous size of the wheat genome.Recent advances in genome sequencing and sequence assembly have produced a high-quality genome sequence for wheat.Here,we suggest that the strategies used to characterize biological mechanisms in model species,including mutant preparation and characterization,gene cloning methods,and improved transgenic technology,can be applied to wheat biology.These strategies will accelerate progress in wheat biology and promote wheat breeding program development.We also outline recent advances in wheat functional genomics.Finally,we discuss the future of wheat functional genomics and the rational design-based molecular breeding of new wheat varieties to contribute to world food security.

A functional characterization of TaMs1 orthologs in Poaceae plants

TaMs1 encodes a non-specific lipid transfer protein(nsLTP) and is required for pollen development in wheat. Although MS1 is a Poaceae-specific gene, the roles of MS1 genes in other Poaceae plants are unknown, especially in rice and maize. Here, we identified one ortholog in rice(OsLTPg29) and two orthologs in maize(ZmLTPg11 and ZmLTPx2). Similar to TaMs1, both OsLTPg29 and ZmLTPg11 genes are specifically expressed in the microsporocytes, and both OsLTPg29 and ZmLTPg11 proteins showed lipid-binding ability to phosphatidic acid and several phosphoinositides. To determine their roles in pollen development, we created osltpg29 mutants and zmltpg11 zmltpx2 double mutants by CRISPR/Cas9.osltpg29, not zmltpg11 zmltpx2, is defective in pollen development, and only OsLTPg29, not ZmLTPg11,can rescue the male sterility of tams1 mutant. Our results demonstrate that the biological function of MS1 in pollen development differs in the evolution of Poaceae plants.

CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat

Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops.High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat.The emerging effective genome editing tool,CRISPR/Cas9 system,makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles.To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat,we compared four different RNA polymerase(Pol)Ⅲpromoters(TaU3 p,TaU6 p,OsU3 p,and OsU6 p)and three types of sgRNA scaffold in the protoplast system.We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective.The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles,whose orthologs,OsNP1 in rice and ZmIPE1 in maize,encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility.Triple homozygous mutations in TaNP1 genes result in complete male sterility.We further demonstrated that anyone wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility.Taken together,this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.

The SNW Domain of SKIP Is Required for Its Integration into the Spliceosome and Its Interaction with the Pall Complex in Arabidopsis

SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptional activator by interacting with the Pall complex. In this study, we identified two nuclear localization signals in SKIP and confirmed that each is sufficient to target SKIP to the nucleus. The SNW domain of SKIP is required for both its function as a splicing factor by promoting integration into the spliceosome in response to stress, and its function as a transcriptional activator by controlling its interaction with the Pall complex to participate in flowering. Truncated proteins that included the SNW domain and the N- or C-terminus of SKIP were still able to carry out the functions of the full-length protein in gene splicing and transcriptional activation in Arabidopsis. In addition, we found that SKIP undergoes 26S proteasome-mediated degrada- tion, and that the C-terminus of SKIP is required to maintain the stability of the protein in plant cells. Together, our findings demonstrate the structural domain organization of SKIP and reveal the core domains and motifs underlying SKIP function in plants.

Peptide primary messengers in plants

The peptide primary messengers regulate embryonic development, cell growth and many other activities in animal cells. But recent evidence verified that peptide primary messengers are also involved in plant defense responses, the recognition between pollen and stigma and keep the balance between cell proliferation and differentiations in shoot apical meristems. Those results suggest that plants may actually make wide use of peptide primary messengers, both in embryonic development and late life when they rally their cells to defend against pathogens and insect pests. The recent advance in those aspects is reviewed.

Conservation and divergence of the histone H2B monoubiquitination pathway from yeast to humans and plants

Histone ubiquitination plays a critical role in the regulation of transcription,and histone H2B monoubiquitination(H2Bub1)is mainly associated with transcriptional activation.Recent studies in yeast,humans,and Arabidopsis have revealed the conservation of chromatin modification via H2Bub1 during evolution.Rad6-Bre1 and their homologs are responsible for H2B monoubiquitination in diverse eukaryotic organisms,and the PAF complex is required for H2Bub1 to proceed.H2Bub1 is involved in many developmental processes in yeast,humans,and Arabidopsis,and it activates gene transcription by regulating the H3K4 methylation state.Notably,the level of H3K4 methylation is entirely dependent on H2Bub1 in yeast and humans,whereas the H3K4 methylation level of only a small number of genes in Arabidopsis is dependent on H2Bub1.In this review,we summarize the enzymes involved in H2B monoubiquitination and deubiquitination,and discuss the biologic functions of H2Bub1 in different organisms.In addition,we focus on recent advances in our understanding of the molecular mechanisms that enable H2Bub1 to perform its function.

Activation effect of extracellular calmodulin on heterotrimeric G protein in pollen plasma membrane

IN recent years, calmodulin （CaM）, an important Ca2+ receptor and constituent of cellular signal transduction systems, has been found extracellularly. We have verified that CaM is presented extracellularly in all of plant species we have examined. In addition, we have reported that extracellular CaM has some biological significance, such as stimulation of cell proliferation, cell wall regeneration, initiation of pollen germination and tube growth and inducement of rbcS gene expression. The role of heterotrimeric G proteins in pollen germination, tube growth and signal transduction of extracellular CaM has been examined in Lily pollen, and two kinds of antibodies against animal Gzα internal sequence and N-terminal

SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis

Deciphering the mechanisms underlying plant responses to abiotic stress is key for improving plant stress resistance. Much is known about the regulation of gene expression in response to salt stress at the tran- scriptional level; however, little is known about this process at the posttranscriptional level. Recently, we demonstrated that SKIP is a component of spliceosome that interacts with clock gene pre-mRNAs and is essential for regulating their alternative splicing and mRNA maturation. In this study, we found that skip-1 plants are hypersensitive to both salt and osmotic stresses, and that SKIP is required for the alter- native splicing and mRNA maturation of several salt-tolerance genes, including NHXl, CBL1, P5CS1, RCl2A, and PATIO. A genome-wide analysis revealed that SKIP mediates the alternative splicing of many genes under salt-stress conditions, and that most of the alternative splicing events in skip-1 involve intron retention and can generate a premature termination codon in the transcribed mRNA. SKIP also controls alternative splicing by modulating the recognition or cleavage of 5＇ and 3＇ splice donor and acceptor sites under salt-stress conditions. Therefore, this study addresses the fundamental question of how the mRNA splicing machinery in plants contributes to salt-stress responses at the posttranscriptional level, and provides a link between alternative splicing and salt tolerance.

A transceptor-channel complex couples nitrate sensing to calcium signaling in Ambidopsis

Nitrate-induced Ca^(2+) signaling is crucial for the primary nitrate response in plants.However,the molecular mechanism underlying the generation of the nitrate-specific calcium signature remains unknown.We report here that a cyclic nucleotide-gated channel(CNGC)protein,CNGC15,and the nitrate transceptor(NRT1.1)constitute a molecular switch that controls calcium influx depending on nitrate levels.The expression of CNGC15 is induced by nitrate,and its protein is localized at the plasma membrane after establishment of young seedlings.We found that disruption of CNGC15 results in the loss of the nitrate-induced Ca^(2+) signature(primary nitrate response)and retards root growth,reminiscent of the phenotype observed in the nrt1.1 mutant.We further showed that CNGC15 is an active Ca^(2+)-permeable channel that physically interacts with the NRT1.1 protein in the plasma membrane.Importantly,we discovered that CNGC15-NRT1.1 interaction silences the channel activity of the heterocomplex,which dissociates upon a rise in nitrate levels,leading to reactivation of the CNGC15 channel.The dynamic interactions between CNGC15 and NRT1.1 therefore control the channel activity and Ca^(2+) influx in a nitrate-dependent manner.Our study reveals a new nutrient-sensing mechanism that utilizes a nutrient transceptor-channel complex assembly to couple nutrient status to a specific Ca^(2+) signature.

Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding

Common wheat(Triticum aestivum L.)is one of the three major food crops in the world;thus,wheat breeding programs are important for world food security.Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding.Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools,especially CRISPR/Cas9 genome editing technology,which allows multiplex genome engineering.Here,we describe the development of CRISPR/Cas9,which has revo-lutionized the field of genome editing.In addition,we emphasize technological breakthroughs(e.g.base editing and prime editing)based on CRISPR/Cas9.We also summarize recent applications and advances in the functional annotation and breeding of wheat,and we introduce the production of CRISPR-edited DNA-free wheat.Combined with other achievements,CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

A chloride efflux transporter,BIG RICE GRAIN 1,is involved in mediating grain size and salt tolerance in rice

Grain size is determined by the size and number of cells in the grain.The regulation of grain size is crucial for improving crop yield;however,the genes and molecular mechanisms that control grain size remain elusive.Here,we report that a member of the detoxification efflux carrier/Multidrug and Toxic Compound Extrusion(DTX/MATE)family transporters,BIG RICE GRAIN 1(BIRG1),negatively influences grain size in rice(Oryza sativa L.).BIRG1 is highly expressed in reproductive organs and roots.In birg1 grain,the outer parenchyma layer cells of spikelet hulls are larger than in wild-type(WT)grains,but the cell number is unaltered.When expressed in Xenopus laevis oocytes,BIRG1 exhibits chloride efflux activity.Consistent with this role of BIRG1,the birg1 mutant shows reduced tolerance to salt stress at a toxic chloride level.Moreover,grains from birg1 plants contain a higher level of chloride than those of WT plants when grown under normal paddy field conditions,and the roots of birg1 accumulate more chloride than those of WT under saline conditions.Collectively,the data suggest that BIRG1 in rice functions as a chloride efflux transporter that is involved in mediating grain size and salt tolerance by controlling chloride homeostasis.

An expression atlas of miRNAs in Arabidopsis thaliana

MicroRNAs(miRNAs) are small non-coding RNAs that regulate a variety of biological processes. miRNA expression often exhibits spatial and temporal specificity. However, genome-wide miRNA expression patterns in different organs during development of Arabidopsis thaliana have not yet been systemically investigated. In this study, we sequenced small RNA libraries generated from 27 different organ/tissue types, which cover the entire life cycle of Arabidopsis. Analysis of the sequencing data revealed that most miRNAs are ubiquitously expressed, whereas a small set of miRNAs display highly specific expression patterns. In addition, different miRNA members within the same family have distinct spatial and temporal expression patterns. Moreover, we found that some miRNAs are produced from different arms of their hairpin precursors at different developmental stages. This work provides new insights into the regulation of miRNA biogenesis and a rich resource for future investigation of miRNA functions in Arabidopsis.

The Arabidopsis AGAMOUS 5′-UTR represses downstream gene translation

Dear Editor,In eukaryotes, mature m RNAs have a tripartite structure consisting of a 5′-untranslated region(5′-UTR), a coding region and a 3′-untranslated region(3′-UTR). Though the coding region encodes the protein sequence.

MicroRNAs and their targets from Arabidopsis to rice: half conserved and half diverged

MicroRNAs are 21-to 24-nucleotide long,endogenous non-coding RNAs in eukaryotes(Hannon,2002).Mature microRNAs generated by Dicer are incorporated into an RNA-induced silencing complex(RISC),resulting in gene silencing via the cleavage of a target mRNA or the repression of target mRNA translation(Qi et al,2005).Thus,microRNAs play a key role in post-transcriptional gene silencing,and microRNA-based gene silencing pathways are critical in developmental regulation,biotic and abiotic adaptations,and hormone responses in plants(Baulcombe,2004;Jones-Rhoades et al.,2006).

Effects of extracellular calmodulin on pollen germination and tube growth

The effects of calmodulin (CaM) antagonist W7_agarose, anti_CaM serum and exogenous purified CaM on pollen germination and tube growth of Forsythia suspensa were studied. The pollen germination and tube growth were inhibited or completely stopped by CaM antagonist W7_agarose. The addition of exogenous purified CaM stimulated pollen germination and tube growth, whereas the same amount of bovine serum albumin (BSA) had no effect. The inhibitory effects caused by W7_agarose and anti_CaM serum could be reversed completely by the addition of exogenous purified CaM. The tube growth of germinated pollen was also inhibited or completely stopped by W7_agarose. The results suggest that endogenous extracellular CaM initiates pollen germination and tube growth, whereas exogenous CaM enhances the above processes.