Cassava Me RS40 is required for the regulation of plant salt tolerance

Soil salinity affects the expression of serine/arginine-rich(SR) genes and isoforms by alternative splicing, which in turn regulates the adaptation of plants to stress.We previously identified the cassava spliceosomal component 35 like(SCL) and SR subfamilies, belonging to the SR protein family, which are extensively involved in responses to abiotic stresses.However, the post-transcriptional regulatory mechanism of cassava arginine/serine-rich(RS) subfamily in response to salt stress remains to be explored.In the current study, we identified 37 genes of the RS subfamily from 11 plant species and systematically investigated the transcript levels of the RS40 and RS31 genes under diverse abiotic stress conditions.Subsequently, an analysis of the conserved protein domains revealed that plant RS subfamily genes were likely to preserve their conserved molecular functions and played critical functional roles in responses to abiotic stresses.Importantly, we found that overexpression of MeRS40 in Arabidopsis enhanced salt tolerance by maintaining reactive oxygen species homeostasis and up-regulating the salt-responsive genes.However,overexpression of MeRS40 gene in cassava reduced salt tolerance due to the depression of its endogenous gene expression by negative autoregulation of its own pre-mRNA.Moreover, the MeRS40 protein interacted with MeU1-70Ks(MeU1-70Ka and MeU1-70Kb) in vivo and in vitro, respectively.Therefore, our findings highlight the critical role of cassava SR proteins in responses to salt stress in plants.

Global characterization of OsPIP aquaporins reveals that the H_(2)O_(2)transporter OsPIP2;6 increases resistance to rice blast

Plasma membrane intrinsic proteins(PIPs)are conserved plant aquaporins that transport small molecules across the plasma membrane to trigger instant stress responses and maintain cellular homeostasis under biotic and abiotic stress.To elucidate their roles in plant immunity to pathogen attack,we characterized the expression patterns,subcellular localizations,and H_(2)O_(2)-transport ability of 11 OsPIPs in rice(Oryza sativa),and identified OsPIP2;6 as necessary for rice disease resistance.OsPIP2;6 resides on the plasma membrane and facilitates cytoplasmic import of the immune signaling molecule H_(2)O_(2).Knockout of OsPIP2;6 increases rice susceptibility to Magnaporthe oryzae,indicating a positive function in plant immunity.OsPIP2;6 interacts with OsPIP2;2,which has been reported to increase rice resistance to pathogens via H_(2)O_(2)transport.Our findings suggest that OsPIP2;6 cooperates with OsPIP2;2 as a defense signal transporter complex during plant–pathogen interaction.

Negative feedback regulation of PROG1 in rice

Cultivated rice(Oryza sativa L.),one of the most widely grown and important crops worldwide,is believed to have been domesticated from the ancestral wild rice(Oryza rufipogon Griff.)thousands of years ago(Cheng et al.2003;Khush 1997;Fuller et al.2010).

Molecular regulation and genetic control of rice thermal response

Global warming threatens food security.Rice(Oryza sativa L.),a vital food crop,is vulnerable to heat stress,especially at the reproductive stage.Here we summarize putative mechanisms of high-temperature perception(via RNA secondary structure,the phyB gene,and phase separation)and response(membrane fluidity,heat shock factors,heat shock proteins,and ROS(reactive oxygen species)scavenging)in plants.We describe how rice responds to heat stress at different cell-component levels(membrane,endoplasmic reticulum,chloroplasts,and mitochondria)and functional levels(denatured protein elimination,ROS scavenging,stabilization of DNA and RNA,translation,and metabolic flux changes).We list temperature-sensitive genetic male sterility loci available for use in rice hybrid breeding and explain the regulatory mechanisms associated with some of them.Breeding thermotolerant rice species without yield penalties via natural alleles mining and transgenic editing should be the focus of future work.

Plant regeneration via protoplast electrofusion in cassava

Protoplast electrofusion between callus protoplasts of cultivar TMS60444 and mesophyll protoplasts of cultivar SC8 was performed as an approach for the genetic improvement of cassava.The fusion products were subsequently cultured in protoplast culture medium(TM2 G) with gradual dilution for approximately 1-2 months.Then the protoplast-derived compact calli were transferred to suspension culture medium(SH) for suspension culture.The cultured products developed successively into embryos,mature embryos,and shoots on somatic embryo emerging medium(MSN),embryo maturation medium(CMM),and shoot elongation medium(CEM),respectively.And the shoots were then rooted on Murashige and Skoog(1962) medium(MS).Sixty-six cell lines were obtained and 12 of them developed into plantlets.Based on assessment of ploidy level and chromosome counting,four of these plantlets were tetraploid and the remaining eight were diploid.Based on assessment of ploidy level and simple sequence repeat(SSR) analysis,nine tetraploid cell lines,one diploid variant plant line and nine variant cell lines were obtained.The diploid variant plant line and the nine variant cell lines all showed partial loss of genetic material compared to that of the parent TMS60444,based on SSR patterns.These results showed that some new germplasm of cassava were created.In this study,a protocol for protoplast electrofusion was developed and validated.Another important conclusion from this work is the confirmation of a viable protocol for the regeneration of plants from cassava protoplasts.Going forward,we hope to provide technical guidance for cassava tissue culture,and also provide some useful inspiration and reference for further genetic improvement of cassava.

MORN motifs in plant PIPKs are involved in the regulation of subcellular localization and phospholipid binding

Multiple repeats of membrane occupation and recognition nexus (MORN) motifs were detected in plant phosphatidylinositl monophosphate kinase (PIPK), a key enzyme in PI-signaling pathway. Structural analysis indicates that all the MORN motifs (with varied numbers at ranges of 7-9), which shared high homologies to those of animal ones, were located at N-terminus and sequentially arranged, except those of OsPIPK1 and AtPIPK7, in which the last MORN motif was separated others by an -100 amino-acid "island" region, revealing the presence of two kinds of MORN arrangements in plant PIPKs. Through employing a yeast-based SMET (sequence of membrane-targeting) system, the MORN motifs were shown being able to target the fusion proteins to cell plasma membrane, which were further confirmed by expression of fused MORN-GFP proteins. Further detailed analysis via deletion studies indicated the MORN motifs in OsPIPK 1, together with the 104 amino-acid "island" region are involved in the regulation of differential subcellular localization, i.e. plasma membrane or nucleus, of the fused proteins. Fat Western blot analysis of the recombinant MORN polypeptide, expressed in Escherichia coli, showed that MORN motifs could strongly bind to PA and relatively slightly to PI4P and PI(4,5)P2. These results provide informative hints on mechanisms of subcellular localization, as well as regulation of substrate binding, of plant PIPKs.

Molecular understanding of wood formation in trees

Trees convert and store the majority of their photosynthetic products in wood which is an essential renewable resource much in demand by human society.Formation of wood follows a process of consecutive cell developmental stages,from vascular cambium proliferation,cell expansion and differentiation,secondary cell wall deposition to programmed cell death,which is controlled by the functionality of complex molecular networks.What are the molecular networks involved in wood formation?How do the molecular networks act in a way to generate wood tissue during tree growth?What are the regulatory modules that lead to the formation of various wood characteristics?The answers to these questions are fundamental to understanding how trees grow,as well as how we can genetically engineer trees with desired properties of wood for human needs.In recent years,a great deal of interest has been invested in the elucidation of wood formation at the molecular level.This review summarizes the current state of understanding of the molecular process that guides wood formation in trees.

Down-regulation of MeMYB2 leads to anthocyanin accumulation and increases chilling tolerance in cassava(Manihot esculenta Crantz)

Chilling-induced accumulation of reactive oxygen species(ROS) is harmful to plants,which usually produce anthocyanins to scavenge ROS as protection from chilling stress.As a tropical crop,cassava is hypersensitive to chilling,but the biochemical basis of this hypersensitivity remains unclear.We previously generated Me MYB2-RNAi transgenic cassava with increased chilling tolerance.Here we report that Me MYB2-RNAi transgenic cassava accumulated less ROS but more cyanidin-3-O-glucoside than the wild type under early chilling stress.Under this stress,the anthocyanin biosynthesis pathway was more active in Me MYB2-RNAi lines than in the wild type,and several genes involved in the pathway,including Me TT8,were up-regulated by Me MYB2-RNAi in the transgenic cassava.Me MYB2 bound to the Me TT8 promoter and blocked its expression under both normal and chilling conditions,thereby inhibiting anthocyanin accumulation.Me TT8 was shown to bind to the promoter of Dihydroflavonol 4-reductase(Me DFR-2)and increased Me DFR-2 expression.Me MYB2 appears to act as an inhibitor of chilling-induced anthocyanin accumulation during the rapid response of cassava to chilling stress.

Wood of trees:Cellular structure,molecular formation,and genetic engineering

Wood is an invaluable asset to human society due to its renewable nature,making it suitable for both sustainable energy production and material manufacturing.Additionally,wood derived from forest trees plays a crucial role in sequestering a significant portion of the carbon dioxide fixed during photosynthesis by terrestrial plants.Nevertheless,with the expansion of the global population and ongoing industrialization,forest coverage has been substantially decreased,resulting in significant challenges for wood production and supply.Wood production practices have changed away from natural forests toward plantation forests.Thus,understanding the underlying genetic mechanisms of wood formation is the foundation for developing high-quality,fast-growing plantation trees.Breeding ideal forest trees for wood production using genetic technologies has attracted the interest of many.Tremendous studies have been carried out in recent years on the molecular,genetic,and cell-biological mechanisms of wood formation,and considerable progress and findings have been achieved.These studies and findings indicate enormous possibilities and prospects for tree improvement.This review will outline and assess the cellular and molecular mechanisms of wood formation,as well as studies on genetically improving forest trees,and address future development prospects.

The molecular basis of heat stress responses in plants

Global warming impacts crop production and threatens food security.Elevated temperatures are sensed by different cell components.Temperature increases are classified as either mild warm temperatures or excessively hot temperatures,which are perceived by distinct signaling pathways in plants.Warm temperatures induce thermomorphogenesis,while high-temperature stress triggers heat acclimation and has destructive effects on plant growth and development.In this review,we systematically summarize the heat-responsive genetic networks in Arabidopsis and crop plants based on recent studies.In addition,we highlight the strategies used to improve grain yield under heat stress from a source-sink perspective.We also discuss the remaining issues regarding the characteristics of thermosensors and the urgency required to explore the basis of acclimation under multifactorial stress combination.

Plant regeneration in the new era:from molecular mechanisms to biotechnology applications

Plants or tissues can be regenerated through various pathways.Like animal regeneration,cell totipotency and pluripotency are the molecular basis of plant regeneration.Detailed systematic studies on Arabidopsis thaliana gradually unravel the fundamental mechanisms and principles underlying plant regeneration.Specifically,plant hormones,cell division,epigenetic remodeling,and transcription factors play crucial roles in reprogramming somatic cells and reestablishing meristematic cells.Recent research on basal non-vascular plants and monocot crops has revealed that plant regeneration differs among species,with various plant species using distinct mechanisms and displaying significant differences in regenerative capacity.Conducting multi-omics studies at the single-cell level,tracking plant regeneration processes in real-time,and deciphering the natural variation in regenerative capacity will ultimately help understand the essence of plant regeneration,improve crop regeneration efficiency,and contribute to future crop design.

Rice leaf inclination2, a VIN3-1ike protein, regulates leaf angle through modulating cell division of the collar

As an important agronomic trait, inclination of leaves is crucial Ior crop architecture and grain yields. 10 understand the molecular mechanism controlling rice leaf angles, one rice leaf inclination2 （1c2, three alleles） mutant was identified and functionally characterized. Compared to wild-type plants, lc2 mutants have enlarged leaf angles due to increased cell division in the adaxial epidermis of lamina joint. The LC2 gene was isolated through positional cloning, and encodes a vernalization insensitive 3-like protein. Complementary expression of LC2 reversed the enlarged leaf angles of lc2 plants, confirming its role in controlling leaf inclination. LC2 is mainly expressed in the lamina joint during leaf development, and particularly, is induced by the phytohormones abscisic acid, gibberellic acid, auxin, and brassinosteroids. LC2 is localized in the nucleus and defects of LC2 result in altered expression of cell division and hormone-responsive genes, indicating an important role of LC2 in regulating leaf inclination and mediating hormone effects.

Gene expression and metabolite profiles of cotton fiber during cell elongation and secondary cell wall synthesis

Cotton fibers elongate rapidly after initiation of elongation, eventually leading to the deposit of a large amount of cellulose. To reveal features of cotton fiber cells at the fast elongation and the secondary cell wall synthesis stages, we compared the respective transcriptomes and metabolite profiles. Comparative analysis of transcriptomes by cDNA array identified 633 genes that were differentially regulated during fiber development. Principal component analysis （PCA） using expressed genes as variables divided fiber samples into four groups, which are diagnostic of developmental stages. Similar grouping results are also found if we use non-polar or polar metabolites as variables for PCA of developing fibers. Auxin signaling, wall-loosening and lipid metabolism are highly active during fiber elongation, whereas cellulose biosynthesis is predominant and many other metabolic pathways are downregulated at the secondary cell wall synthesis stage. Transcript and metabolite profiles and enzyme activities are consistent in demonstrating a specialization process of cotton fiber development toward cellulose synthesis. These data demonstrate that cotton fiber cell at a certain stage has its own unique feature, and developmental stages of cotton fiber cells can be distinguished by their transcript and metabolite profiles. During the secondary cell wall synthesis stage, metabolic pathways are streamed into cellulose synthesis.

Reduction in cadmium accumulation in japonica rice grains by CRISPR/Cas9-mediated editing of OsNRAMP5

Cadmium(Cd) intake is harmful to human health and Cd contamination in rice grains represents a severe threat to those consuming rice as a staple food. Knockout of Cd transporters is a promising strategy to reduce Cd accumulation in rice grains. OsNRAMP5 is the major transporter for Cd and manganese(Mn) uptake in rice. Nevertheless, it is uncertain whether knockout of OsNRAMP5 is applicable to produce low Cd rice without affecting plant growth and grain yield. In this study, we adopted CRISPR/Cas9-based gene editing technology to knock out OsNRAMP5 in two japonica varieties. We generated three independent transgene-free osnramp5 mutants and investigated the effect of osnramp5 mutations on Cd accumulation and plant growth. Hydroponic experiments showed that plant growth and chlorophyll content were significantly reduced in osnramp5 mutants at low Mn conditions, and this defective growth in the mutants could be fully rescued by supply of high levels of Mn. Cd and Mn accumulation in both roots and shoots was markedly reduced in the mutants compared to that in wild-type plants. In paddy field experiments, although Cd in flag leaves and grains was greatly reduced in osnramp5 mutants, some agronomic traits including plant height, seed setting rate, and grain number per panicle were affected in the mutants, which ultimately caused a mild reduction in grain yield. The reduced plant growth in the mutants can be attributed to a marked decrease in Mn accumulation. Our results reveal that the manipulation of OsNRAMP5 should be treated with caution: When assessing the applicability of osnramp5 mutants, soil pH and soil water content in paddy fields need to be taken into consideration, since they might affect the levels of available Mn in the soil and consequently determine the effect of the mutation on grain yield.

Gibberellin homeostasis and plant height control by EUI and a role for gibberellin in root gravity responses in rice

The rice Eui （ELONGATED UPPERMOST INTERNODE） gene encodes a cytochrome P450 monooxygenase that deactivates bioactive gibberellins （GAs）. In this study, we investigated controlled expression of the Eui gene and its role in plant development. We found that Eui was differentially induced by exogenous GAs and that the Eui promoter had the highest activity in the vascular bundles. The eui mutant was defective in starch granule development in root caps and Eui overexpression enhanced starch granule generation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Experiments using embryoless half-seeds revealed that RAmylA and GAmyb were highly upregulated in eui aleurone ceils in the absence of exogenous GA. In addition, the GA biosynthesis genes GA3oxl and GA20ox2 were downregulated and GA2oxl was upregulated in eui seedlings. These results indicate that EUI is involved in GA homeostasis, not only in the internodes at the heading stage, but also in the seedling stage, roots and seeds. Disturbing GA homeostasis affected the expression of the GA signaling genes GID1 （GIBBERELLIN INSENSITIVE DWARF 1）, GID2 and SLR1. Transgenic RNA interference of the Eui gene effectively increased plant height and improved heading performance. By contrast, the ectopic expression of Eui under the promoters of the rice GA biosynthesis genes GA3ox2 and GA2Oox2 significantly reduced plant height. These results demonstrate that a slight increase in Eui expression could dramatically change rice morphology, indicating the practical application of the Eui gene in rice molecular breeding for a high yield potential.

Membrane steroid-binding protein 1 （MSBP1） negatively regulates brassinosteroid signaling by enhancing the endocytosis of BAK1

Brassinosteroids （BRs） are perceived by transmembrane receptors and play vital roles in plant growth and development, as well as cell in responses to environmental stimuli. The transmemhrane receptor BRI1 can directly bind to brassinolide （BL）, and BAK1 interacts with BRI1 to enhance the BRI1-mediated BR signaling. Our previous studies indicated that a membrane steroid-binding protein 1 （MSBP1） could bind to BL in vitro and is negatively involved in BR signaling. To further elucidate the underlying mechanism, we here show that MSBPI specifically interacts with the extraeellular domain of BAK1 in vivo in a BL-independent manner. Suppressed cell expansion and BR responses by increased expression of MSBP1 can be recovered by overexpressing BAK1 or its intracellnlar kinase domain, sug- gesting that MSBP1 may suppress BR signaling through interacting with BAK1. Subcellular localization studies re- vealed that both MSBPI and BAK1 are localized to plasma membrane and endocytic vesicles and MSBP1 accelerates BAK1 endocytosis, which results in suppressed BR signaling by shifting the equilibrium of BAKI toward endosomes. Indeed, enhanced MSBP1 expression reduces the interaction between BRI1 and BAK1 in vivo, demonstrating that MSBP1 acts as a negative factor at an early step of the BR signaling pathway.

Genome-wide analysis of the phospholipase D family in Oryza sativa and functional characterization of PLDβ1 in seed germination

Phospholipase D （PLD） plays a critical role in plant growth and development, as well as in hormone and stress responses. PLD encoding genes constitute a large gene family that are present in higher plants. There are 12 members of the PLD family in Arabidopsis thaliana and several of them have been functionally characterized; however, the members of the PLD family in Oryza sativa remain to be fully described. Through genome-wide analysis, 17 PLD members found in different chromosomes have been identified in rice. Protein domain structural analysis reveals a novel subfamily, besides the C2-PLDs and PXPH-PLDs, that is present in rice - the SP-PLD. SP-PLD harbors a signal peptide instead of the C2 or PXPH domains at the N-terminus. Expression pattern analysis indicates that most PLD-encoding genes are differentially expressed in various tissues, or are induced by hormones or stress conditions, suggesting the involvement of PLD in multiple developmental processes. Transgenic studies have shown that the suppressed expression office PLDβ1 results in reduced sensitivity to exogenous ABA during seed germination. Further analysis of the expression of ABA signaling-related genes has revealed that PLDβ1 stimulates ABA signaling by activating SAPK, thus repressing GAmyb exoression and inhibiting seed germination.

Functional conservation of the meiotic genes SDS and RCK in male meiosis in the monocot rice

The Arabidopsis SDS （SOLO DANCERS） and RCK （ROCK-N-ROLLERS） genes are important for male meiosis, but it is still unknown whether they represent conserved functions in plants. We have performed phylogenetic analyses of SDS and RCK and their respective homologs, and identified their putative orthologs in poplar and rice. Quantitative real-time RT-PCR analysis indicated that rice SDS and RCK are expressed preferentially in young flowers, and transgenic RNAi rice lines with reduced expression of these genes exhibited normal vegetative development, but showed significantly reduced fertility with partially sterile flowers and defective pollens. SDS deficiency also caused a decrease in pollen amounts. Further cytological examination of male meiocytes revealed that the SDS deficiency led to defects in homolog interaction and bivalent formation in meiotic prophase I, and RCK deficiency resulted in defective meiotic crossover formation. These results indicate that rice SDS and RCK genes have similar functions to their Arabidopsis orthologs. Because rice and Arabidopsis, respectively, are members of monocots and eudicots, two largest groups of flowering plants, our results suggest that the functions of SDS and RCK are likely conserved in flowering plants.

Studies on rice seed quality through analysis of a large- scale T-DNA insertion population

A rice （Oryza sativa） T-DNA insertion population, which included more than 63 000 independent transgenic lines and 8 840 identified flanking sequence tags （FSTs） that were mapped onto the rice genome, was developed to systemi- cally study the rice seed quality control. Genome-wide analysis of the FST distribution showed that T-DNA insertions were positively correlated with expressed genes, but negatively with transposable elements and small RNAs. In addition, the recovered T-DNAs were preferentially located at the untranslated region of the expressed genes. More than 11 000 putative homozygous lines were obtained through multi-generations of planting and resistance screening, and measurement of seed quality of around half of them, including the contents of starch, amylose, protein and fat, with a nondestructive near-infrared spectroscopy method, identified 551 mutants with unique or multiple altered param- eters of seed quality. Analysis of the corresponding FSTs showed that genes participating in diverse functions, including metabolic processes and transcriptional regulation, were involved, indicating that seed quality is regulated by a complex network.

The role of Arabidopsis 5PTase13 in root gravitropism through modulation of vesicle trafficking

Inositol polyphosphate 5-phosphatases （5PTases） are enzymes of phosphatidylinositol metabolism that affect various aspects of plant growth and development. Arabidopsis 5PTasel3 regulates auxin homeostasis and hormonerelated cotyledon vein development, and here we demonstrate that its knockout mutant 5pt13 has elevated sensitivity to gravistimulation in root gravitropic responses. The altered responses of 5pt13 mutants to 1-N-naphthylphthalamic acid （an auxin transport inhibitor） indicate that 5PTase13 might be involved in the regulation of auxin transport. Indeed, the auxin efflux carrier PIN2 is expressed more broadly under 5PTasel3 deficiency, and observations of the internalization of the membrane-selective dye FM4-64 reveal altered vesicle trafficking in 5pt13 mutants. Compared with wild-type, 5pt13 mutant seedlings are less sensitive to the inhibition by brefeldin A of vesicle cycling, seedling growth, and the intracellular cycling of the PIN1 and PIN2 proteins. Further, auxin redistribution upon gravitropic stimulation is stimulated under 5PTasel3 deficiency. These results suggest that 5PTasel3 may modulate auxin transport by regulating vesicle trafficking and thereby play a role in root gravitropism.