Identification and characterization of the chalkiness endosperm gene CHALK-H in rice(Oryza sativa L.)

Chalkiness is one of the most important agronomic traits in rice breeding,which directly affects the quality of rice seed.In this study,we identified a chalkiness endosperm mutant,chalk-h,from N-methyl-N-nitrosourea(MNU)-induced japonica rice cultivar Hwacheong(HC).Compared with wild type(WT)-HC,chalk-h showed severe chalkiness in the endosperm,yellowish green leaves,as well as reduced plant height.Scanning electron microscopy(SEM)analysis showed that starch grains in the chalk-h mutant were irregular in size and loosely arranged,with large gaps between granules,forming ovoid or orbicular shapes.MutMap analysis revealed that the phenotype of chalk-h is controlled by a single recessive gene LOC_Os11g39670 encoding seryl-tRNA synthetase,which is renamed as CHALK-H.A point mutation occurs in chalk-h on the sixth exon(at nucleotide 791)of CHALK-H,in which adenine(A)is replaced by thymidine(T),resulting in an amino acid codon change from glutamine(Glu)to valine(Val).The chalk-h mutant exhibited a heat-sensitive phenotype from the 3-leaf stage,including yellow-green leaves and reduced pigment content.The transcriptional expression of starch synthesis-related genes was down-regulated in the chalk-h mutants compared to WT-HC at different grain-filling stages.With an increase in temperature,the expression of photosynthesis-related genes was down-regulated in the chalk-h mutant compared to WT-HC.Overexpression of CHALK-H rescued the phenotype of chalk-h,with endosperm and leaf color similar to those of WT-HC.Our findings reveal that CHALK-H is a causative gene controlling chalkiness and leaf color of the chalk-h mutant.CHALK-H is the same gene locus as TSCD11,which was reported to be involved in chloroplast development under high temperature.We suggest that CHALK-H/TSCD11 plays important roles not only in chloroplast development,but also in photosynthesis and starch synthesis during rice growth and development,so it has great application potential in rice breeding for high quality and yield.

Low temperature-mediated repression and far-red light-mediated induction determine morning FLOWERING LOCUS T expression levels

In order to flower in the appropriate season,plants monitor light and temperature changes and alter downstream pathways that regulate florigen genes such as Arabidopsis(Arabidopsis thaliana)FLOWERING LOCUS T(FT).In Arabidopsis,FT messenger RNA levels peak in the morning and evening under natural long-day conditions(LDs).However,the regulatory mechanisms governing morning FT induction remain poorly understood.The morning FT peak is absent in typical laboratory LDs characterized by high red:far-red light(R:FR)ratios and constant temperatures.Here,we demonstrate that ZEITLUPE(ZTL)interacts with the FT repressors TARGET OF EATs(TOEs),thereby repressing morning FT expression in natural environments.Under LDs with simulated sunlight(R:FR=1.0)and daily temperature cycles,which are natural LD-mimicking environmental conditions,FT transcript levels in the ztl mutant were high specifically in the morning,a pattern that was mirrored in the toe1 toe2 double mutant.Low night-to-morning temperatures increased the inhibitory effect of ZTL on morning FT expression by increasing ZTL protein levels early in the morning.Far-red light counteracted ZTL activity by decreasing its abundance(possibly via phytochrome A(phyA))while increasing GIGANTEA(GI)levels and negatively affecting the formation of the ZTL-GI complex in the morning.Therefore,the phyA-mediated high-irradiance response and GI play pivotal roles in morning FT induction.Our findings suggest that the delicate balance between low temperature-mediated ZTL activity and the far-red light-mediated functions of phyA and GI offers plants flexibility in fine-tuning their flowering time by controlling FT expression in the morning.

The PRR-EC complex and SWR1 chromatin remodeling complex function cooperatively to repress nighttime hypocotyl elongation by modulating PIF4 expression in Arabidopsis

The circadian clock entrained by environmental light-dark cycles enables plants to fine-tune diurnal growth and developmental responses.Here,we show that physical interactions among evening clock components,including PSEUDO-RESPONSE REGULATOR 5(PRR5),TIMING OF CAB EXPRESSION 1(TOC1),and the Evening Complex(EC)component EARLY FLOWERING 3(ELF3),define a diurnal repressive chromatin structure specifically at the PHYTOCHROME-INTERACTING FACTOR 4(PIF4)locus in Arabidopsis.These three clock components act interdependently as well as independently to repress nighttime hypocotyl elongation,as hypocotyl elongation rate dramatically increased specifically at nighttime in the prr5-1 toc1-21 elf3-1 mutant,concomitantly with a substantial increase in PIF4 expression.Transcriptional repression of PIF4 by ELF3,PRR5,and TOC1 is mediated by the SWI2/SNF2-RELATED(SWR1)chromatin remodeling complex,which incorporates histone H2A.Z at thePIF4 locus,facilitating robust epigenetic suppression ofPIF4 during the evening.Overall,these findings demonstrate that the PRR-EC-SWR1 complex represses hypocotyl elongation at night through a distinctive chromatin domain covering PIF4 chromatin.

ESR2–HDA6 complex negatively regulates auxin biosynthesis to delay callus initiation in Arabidopsis leaf explants during tissue culture

Plants exhibit an astonishing ability to regulate organ regeneration upon wounding.Excision of leaf explants promotes the biosynthesis of indole-3-acetic acid(IAA),which is polar-transported to excised regions,where cell fate transition leads to root founder cell specification to induce de novo root regeneration.The regeneration capacity of plants has been utilized to develop in vitro tissue culture technologies.Here,we report that IAA accumulation near the wounded site of leaf explants is essential for callus formation on 2,4-dichlorophenoxyacetic acid(2,4-D)-rich callus-inducing medium(CIM).Notably,a high concentration of 2,4-D does not compensate for the action of IAA because of its limited efflux;rather,it lowers IAA biosynthesis via a negative feedback mechanism at an early stage of in vitro tissue culture,delaying callus initiation.The auxin negative feedback loop in CIM-cultured leaf explants is mediated by an auxin-inducible APETALA2 transcription factor,ENHANCER OF SHOOT REGENERATION 2(ESR2),along with its interacting partner HISTONE DEACETYLASE 6(HDA6).The ESR2–HDA6 complex binds directly to,and removes the H3ac mark from,the YUCCA1(YUC1),YUC7,and YUC9 loci,consequently repressing auxin biosynthesis and inhibiting cell fate transition on 2,4-D-rich CIM.These findings indicate that negative feedback regulation of auxin biosynthesis by ESR2 and HDA6 interferes with proper cell fate transition and callus initiation.

Analysis of simple sequence repeats in rice bean(Vigna umbellata) using an SSR-enriched library

Rice bean(Vigna umbellata Thunb.), a warm-season annual legume, is grown in Asia mainly for dried grain or fodder and plays an important role in human and animal nutrition because the grains are rich in protein and some essential fatty acids and minerals. With the aim of expediting the genetic improvement of rice bean, we initiated a project to develop genomic resources and tools for molecular breeding in this little-known but important crop.Here we report the construction of an SSR-enriched genomic library from DNA extracted from pooled young leaf tissues of 22 rice bean genotypes and developing SSR markers.In 433,562 reads generated by a Roche 454 GS-FLX sequencer, we identified 261,458 SSRs, of which 48.8% were of compound form. Dinucleotide repeats were predominant with an absolute proportion of 81.6%, followed by trinucleotides(17.8%). Other types together accounted for 0.6%. The motif AC/GT accounted for 77.7% of the total, followed by AAG/CTT(14.3%), and all others accounted for 12.0%. Among the flanking sequences, 2928 matched putative genes or gene models in the protein database of Arabidopsis thaliana, corresponding with 608 non-redundant Gene Ontology terms. Of these sequences, 11.2% were involved in cellular components, 24.2% were involved molecular functions, and 64.6% were associated with biological processes. Based on homolog analysis, 1595 flanking sequences were similar to mung bean and 500 to common bean genomic sequences. Comparative mapping was conducted using 350 sequences homologous to both mung bean and common bean sequences. Finally, a set of primer pairs were designed, and a validation test showed that58 of 220 new primers can be used in rice bean and 53 can be transferred to mung bean.However, only 11 were polymorphic when tested on 32 rice bean varieties. We propose that this study lays the groundwork for developing novel SSR markers and will enhance the mapping of qualitative and quantitative traits and marker-assisted selection in rice bean and other Vigna species.

基于辣椒基因组测序探究辣味在种群中的进化

辣椒(Capsicum annuum)是美洲最古老的驯化作物之一,在全世界被广泛种植。本研究对CM334(墨西哥地方品种)进行了全基因组测序和组装(覆盖度为186.6×),同时还对另外两个栽培种和一个野生种分别进行了重测序和从头(de novo)测序。结果表明辣椒基因组大小约为番茄的4倍,辣椒基因组上存在大量gypsy转座子和花椰菜病毒家族元件的积累。结合转录组综合分析认为,辣椒素合成酶基因表达模式的改变及新功能化是导致辣椒素生物合成的原因,研究还发现辣椒与番茄在乙烯合成及果实成熟调控方面存在明显不同的分子模式。辣椒参考基因组的发布为辣椒营养品质和药用价值的改善提供了重要平台。

Adenosine monophosphate enhances callus regeneration competence for de novo plant organogenesis

Dear Editor,Plant tissue culture involves callus formation and de novo shoot regeneration.First,explants from differentiated tissues are used to generate a pluripotent cell mass,called callus,on auxin-rich callus-inducing medium(CIM),followed by shoot regeneration on cytokinin-rich shoot-inducing medium(SiM).Callus results from division of pericycle-like cells(Atta et al.,2009;Sugimoto et al.,2010);its cellular identity resembles that of lateral root primordia(Atta et al.,2009;Sugimoto et al.,2010).Callus acquires cellular pluripotency by forming root stem cell niches on CIM(Sugimoto et al.,2010)。

Callus proliferation-induced hypoxic microenvironment decreases shoot regeneration competence in Arabidopsis

Plants are aerobic organisms that rely on molecular oxygen for respiratory energy production.Hypoxic conditions,with oxygen levels ranging between 1%and 5%,usually limit aerobic respiration and affect plant growth and development.Here,we demonstrate that the hypoxic microenvironment induced by active cell proliferation during the two-step plant regeneration process intrinsically represses the regener-ation competence of the callus in Arabidopsis thaliana.We showed that hypoxia-repressed plant regener-ation is mediated by the RELATED TO APETALA2.12(RAP2.12)protein,a memberof the Ethylene Response Factor VIl(ERF-Vll)family.We found that the hypoxia-activated RAP2.12 protein promotes salicylic acid(SA)biosynthesis and defense responses,thereby inhibiting pluripotency acquisition and de novo shoot regeneration in calli.Molecular and genetic analyses revealed that RAP2.12 could bind directly to the SALICYLIC ACID INDUCTION DEFICIENT 2(SID2)gene promoter and activate SA biosynthesis,repressing plant regeneration possibly via a PLETHORA(PLT)-dependent pathway.Consistently,the rap2.12 mutant calli exhibits enhanced shoot regeneration,which is impaired by SA treatment.Taken together,these find-ings uncover that the cell proliferation-dependent hypoxic microenvironment reduces cellular pluripotency and plant regeneration through the RAP2.12-SID2 module.

Heat-induced leaf epidermal cell damage triggers autophagy-mediated mesophyll cell expansion in Arabidopsis

Dear Editor,Plants have evolved intricate mechanisms to recognize fluctuating environments,relay signals,and ultimately recover from the cellular and tissue damage imposed by environmental stresses.Upon wounding,mechanical damage is recognized via cellular compounds released from damaged cells(Vega-Munoz et al.,2020)or changes in turgor pressure and cell-wall properties(Hoermayer et al.,2020).

SPL3/4/5 Integrate Developmental Aging and Photoperiodic Signals into the FT-FD Module in Arabidopsis Flowering

Environmental sensitivity varies across developmental phases in flowering plants. In the juvenile phase, mi- croRNA156 （miR156）-mediated repression of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE （SPL） transcription factors renders Arabidopsis plants incompetent to floral inductive signals, including long- day （LD） photoperiod. During the vegetative phase transition, which accompanies a reduction of miR156 and a concomitant elevation of its targets, plants acquire reproductive competence such that LD signals promote flowering. However, it remains largely unknown how developmental signals are associated with photoperiodic flowering. Here, we show that SPL3, SPL4, and SPL5 （SPL3/4/5） potentiate the FLOWERING LOCUS T （FT）-FD module in photoperiodic flowering. SPL3/4/5 function as transcriptional activators through the interaction with FD, a basic leucine zipper transcription factor which plays a critical role in photoperiodic flowering. SPL3/4/5 can directly bind to the promoters of APETALA1, LEAFY, and FRUITFULL, thus mediating their activation by the FT-FD complex. Our findings demonstrate that SPL3/ 4/5 act synergistically with the FT-FD module to induce flowering under LDs, providing a long-sought mo- lecular knob that links developmental aging and photoperiodic flowering.

H3K36me2 is highly correlated with m6A modifications in plants

Various chemical modifications at the nucleosome,including DNA methylation,histone H3 acetylation(H3Ac),and histone H3 mono-/di-/trimethylation at various lysine residues(H3K4me3,H3K9me1/me2,H3K27me3,and H3K36me2/me3),underlie the delicate control of chromatin architecture(Ramirez-Prado et al.2018).These modifications influence gene ex-pression by changing the accessibility of chromatin by RNA PolⅡand chromatin-binding proteins,under-pinning the importance of chromatin landscape in gene regulation(Malapeira et al.2012).

A putative plant organelle RNA recognition protein gene is essential for maize kernel development

Basal endosperm transfer layer（BETL） cells are responsible for transferring apoplastic solutes from the maternal pedicel into the endosperm,supplying the grain with compounds required for embryo development and storage reserve accumulation.Here,we analyze the maize（Zea mays L.） empty pericarp6（emp6） mutant,which causes early arrest in grain development.The Emp6 tgene function is required independently in both the embryo and endosperm.The emp6 mutant causes a notable effect on the differentiation of BETL cells;the extensive cell wall ingrowths that distinguish BETL cells are diminished and BETL marker gene expression is compromised in mutant kernels.Transposon tagging identified the emp6 locus as encoding a putative plant organelle RNA recognition（PORR） protein,1 of 15 PORR family members in maize.The emp6 transcript is widely detected in plant tissues with highest Researclevels in embryos and developing kernels.EMP6-green fluorescent protein（GFP） fusion proteins transiently expressed in Nicotiana benthamiana leaves were targeted specifically to mitochondria.These results suggest that BETL cell differentiation might be particularly energy intensive,or alternatively,that mitochondria might confer a developmental function.

Go green with plant organelle genome editing

Programmable genome-editing technologies that enable efficient and precise genetic manipulation at targeted genomic regions,in eluding zinc fin ger nucleases,tran scripti on activator-like effector nu cleases(TALENs),and clustered regularly in terspaced short palindromic repeat(CRISPR)systems,have become invaluable tools for plant biotech no logy and precision breedi ng(Sedeek et al.,2019).Genome-editing systems have been utilized in a variety of plant species to characterize nuclear gene functions and improve agricultural traits,such as crop yields,nutritional values,and biotic and abiotic stress toleranee(Sedeek et al„2019).

Heat Makes Cellular Hotspots in Plants

Global warming has become the most serious issue in the world.The global average surface temperature has gradually increased over the last 50 years,and unprecedented abnormal climates have occurred around the globe.Ecosystems are being seriously threatened,and many species are endangered due to loss of habitat.Plants are not immune from global warming,but because of their sessile nature,plants have evolved elaborate sensory mechanisms for ambient temperatures.Growing concerns include how plants sense the temperature changes and what natural variations have evolved for plant adaptation to given habitat climates.

GIGANTEA Shapes the Photoperiodic Rhythms of Thermomorphogenic Growth in Arabidopsis

Plants maintain their internal temperature under environments with fluctuating temperatures by adjusting their morphology and architecture,an adaptive process termed thermomorphogenesis.Notably,the rhythmic patterns of plant thermomorphogenesis are governed by day-length information.However,it remains elusive how thermomorphogenic rhythms are regulated by photoperiod.Here,we show that warm temperatures enhance the accumulation of the chaperone GIGANTEA(Gl),which thermostabilizes the DELLA protein,REPRESSOR OF ga1-3(RGA),under long days,thereby attenuating PHYTOCHROME INTERACTING FACTOR 4(PIF4)-mediated thermomorphogenesis.In contrast,under short days,when Gl accumulation is reduced,RGA is readily degraded through the gibberellic acid-mediated ubiquitination-proteasome pathway,promoting thermomorphogenic growth.These data indicate that the GI-RGA-PIF4 signaling module enables plant thermomorphogenic responses to occur in a day-length-dependent manner.We propose that the Gl-mediated integration of photoperiodic and temperature information shapes thermomorphogenic rhythms,which enable plants to adapt to diel fluctuations in day length and temperature during seasonal transitions.

HOS1 Facilitates the Phytochrome B-Mediated Inhibition of PIF4 Function during Hypocotyl Growth in Arabidopsis

Upon exposure to light, developing seedlings undergo photomorphogenesis, as illustrated by inhibition of hypocotyl elongation, cotyledon opening, and leaf greening. During hypocotyl photomorphogenesis, light signals are sensed by multiple photoreceptors, among which the red/far-red light-sensing phytochromes have been extensively studied. However, it is not fully understood how the phytochromes modulate hypo- cotyl growth. Here, we demonstrated that HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 （HOS1）, which is known to either act as E3 ubiquitin ligase or affect chromatin organization, inhibits the transcriptional activation activity of PHYTOCHROME INTERACTING FACTOR 4 （PIF4）, a key transcrip- tion factor that promotes hypocotyl growth. Consistent with the negative regulatory role of HOSl in hypo- cotyl growth, HOSl-defective mutants exhibited elongated hypocotyls in the light. Notably, phyB induces HOS1 activity in inhibiting PIF4 function. Taken together, these observations provide a molecular basis for the phyB-mediated suppression of hypocotyl growth in Arabidopsis.