Honoring a legend: Celebrating the life and legacy of Professor Heng Li

Professor Heng Li(李恒,March 9,1929-January 12,2023)was a legendary plant taxonomist and phytogeographer at the Kunming Institute of Botany,Chinese Academy of Sciences(Fig.1).She made important contributions to our understanding of China's flora,including the biodiversity and biogeography of Dulong Valley and the Gaoligong Mountains,as well as the vegetation of plateau lakes and wetlands in Southwest China.Professor Heng Li received her Bachelor's degree in Russian from Beijing Foreign Studies University in 1956 and immediately joined the Institute of Geography(now Institute of Geographic Sciences and Natural Resources Research),Chinese Academy of Sciences,as a Russian translator.In 1961,Prof.

Monitoring the impact of climate change and human activities on grassland vegetation dynamics in the northeastern Qinghai-Tibet Plateau of China during 2000–2015

Climate change and human activities can influence vegetation net primary productivity(NPP), a key component of natural ecosystems. The Qinghai-Tibet Plateau of China, in spite of its significant natural and cultural values, is one of the most susceptible regions to climate change and human disturbances in the world. To assess the impact of climate change and human activities on vegetation dynamics in the grassland ecosystems of the northeastern Qinghai-Tibet Plateau, we applied a time-series trend analysis to normalized difference vegetation index(NDVI) datasets from 2000 to 2015 and compared these spatiotemporal variations with trends in climatic variables over the same time period. The constrained ordination approach(redundancy analysis) was used to determine which climatic variables or human-related factors mostly influenced the variation of NDVI. Furthermore, in order to determine whether current conservation measures and programs are effective in ecological protection and reconstruction, we divided the northeastern Qinghai-Tibet Plateau into two parts: the Three-River Headwater conservation area(TRH zone) in the south and the non-conservation area(NTRH zone) in the north. The results indicated an overall(73.32%) increasing trend of vegetation NPP in grasslands throughout the study area. During the period 2000–2015, NDVI in the TRH and NTRH zones increased at the rates of 0.0015/a and 0.0020/a, respectively. Specifically, precipitation accounted for 9.2% of the total variation in NDVI, while temperature accounted for 13.4%. In addition, variation in vegetation NPP of grasslands responded not only to long-and short-term changes in climate, as conceptualized in non-equilibrium theory, but also to the impact of human activities and their associated perturbations. The redundancy analysis successfully separated the relative contributions of climate change and human activities, of which village population and agricultural gross domestic product were the two most important contributors to the NDVI changes, explaining 17.8% and 17.1% of the total variation of NDVI(with the total contribution >30.0%), respectively. The total contribution percentages of climate change and human activities to the NDVI variation were 27.5% and 34.9%, respectively, in the northeastern Qinghai-Tibet Plateau. Finally, our study shows that the grassland restoration in the study area was enhanced by protection measures and programs in the TRH zone, which explained 7.6% of the total variation in NDVI.

Mapping and breeding value evaluation of a semi-dominant semidwarf gene in upland rice

Plant height is an important trait related to yield potential and plant architecture.A suitable plant height plays a crucial role in improvement of rice yield and lodging resistance.In this study,we found that the traditional upland landrace 'Kaowenghan'(KWH) showed a special semi-dwarf phenotype.To identify the semi-dwarf gene from KWH,we raised BC2 F4 semi-dwarf introgression lines(IL) by hybridization of the japonica rice cultivar 'Dianjingyou1'(DJY1) and KWH in a DJY1 background.The plant height of the homozygous semi-dwarf IL(IL-87) was significantly reduced compared with that of DJY1.The phenotype of the F1 progeny of the semi-dwarf IL-87 and DJY1 showed that the semi-dwarf phenotype was semidominant.QTL mapping indicated that the semi-dwarf phenotype was controlled by a major QTL q DH1 and was localized between the markers RM6696 and RM12047 on chromosome 1.We also developed near-isogenic lines(NIL) from the BC3 F3 population,and found that the yield of homozygous NIL(NIL-2)was not significantly different compared to DJY1.Breeding value evaluation through investigation of the plant height of the progeny of NIL(NIL-2) and cultivars from different genetic background indicate that the novel semi-dwarf gene shows potential as a genetic resource for rice breeding.

Genome-wide analysis of the B3 transcription factors reveals that RcABI3/VP1 subfamily plays important roles in seed development and oil storage in castor bean(Ricinus communis)

The B3 transcription factors(TFs)in plants play vital roles in numerous biological processes.Although B3 genes have been broadly identified in many plants,little is known about their potential functions in mediating seed development and material accumulation.Castor bean(Ricinus communis)is a non-edible oilseed crop considered an ideal model system for seed biology research.Here,we identified a total of 61 B3 genes in the castor bean genome,which can be classified into five subfamilies,including ABI3/VP1,HSI,ARF,RAV and REM.The expression profiles revealed that RcABI3/VP1 subfamily genes are significantly up-regulated in the middle and later stages of seed development,indicating that these genes may be associated with the accumulation of storage oils.Furthermore,through yeast one-hybrid and tobacco transient expression assays,we detected that ABI3/VP1 subfamily member RcLEC2 directly regulates the transcription of RcOleosin2,which encodes an oil-body structural protein.This finding suggests that RcLEC2,as a seed-specific TF,may be involved in the regulation of storage materials accumulation.This study provides novel insights into the potential roles and molecular basis of B3 family proteins in seed development and material accumulation.

Identification and fine mapping of rtms1-D,a gene responsible for reverse thermosensitive genic male sterility from Diannong S-1X

Thermosensitive genic male sterility(TGMS)has been widely used in two-line hybrid rice breeding.Due to hybrid seed production being highly affected by changeable environments,its application scope is limited to some extent.Thus,it is of great importance to identify potential TGMS genes in specific rice varieties.Here,Diannong S-1 xuan(DNS-1 X),a reverse TGMS(RTGMS)japonica male sterile line,was identified from Diannong S-1.Genetic analysis showed that male sterility was tightly controlled by a single recessive gene,which was supported by the phenotype of the F;and F;populations derived from the cross between DNS-1 X and Yunjing 26(YJ26).Combining simple sequence repeat(SSR)markers and bulked segregation analysis(BSA),we identified a 215 kb region on chromosome 10 as a candidate reverse TGMS region,which was designated as rtmsl-D.It was narrower than the previously reported RTGMS genes rtmsl and tms6(t).The fertility conversion detected in the natural environment showed that DNS-1 X was sterile below 28-30℃;otherwise,it was fertile.Histological analysis further indicated that the pollen abortion was occurred in the young microspore stage.This study will provide new resources for two-line hybrid rice and pave the way for molecular breeding of RTGMS lines.

GhWRKY33 negatively regulates jasmonate-mediated plant defense to Verticillium dahliae

Verticillium wilt,caused by Verticillium dahliae,seriously restricts the yield and quality improvement of cotton.Previous studies have revealed the involvement of WRKY members in plant defense against V.dahliae,but the underlying mechanisms involved need to be further elucidated.Here,we demonstrated that Gossypium hirsutum WRKY DNA-binding protein 33(GhWRKY33) functions as a negative regulator in plant defense against V.dahliae.GhWRKY33 expression is induced rapidly by V.dahliae and methyl jasmonate,and overexpression of GhWRKY33 reduces plant tolerance to V.dahliae in Arabidopsis.Quantitative RT-PCR analysis revealed that expression of several JA-associated genes was significantly repressed in GhWRKY33 overexpressing transgenic plants.Yeast one-hybrid analysis revealed that GhWRKY33 may repress the transcription of both AtERF1 and GhERF2 through its binding to their promoters.Protein-protein interaction analysis suggested that GhWRKY33 interacts with G.hirsutum JASMONATE ZIM-domain protein 3(GhJAZ3).Similarly,overexpression of GhJAZ3 also decreases plant tolerance to V.dahliae.Furthermore,GhJAZ3 acts synergistically with GhWRKY33 to suppress both AtERF1 and GhERF2 expression.Our results imply that GhWRKY33 may negatively regulate plant tolerance to V.dahliae via the JA-mediated signaling pathway.

Different strategies in biomass allocation across elevation in two Gentiana plants on the Yunnan-Guizhou Plateau, China

Patterns of biomass allocation among organs in plants are important because they influence many growth processes.The Yunnan-Guizhou Plateau(YGP)is considered to be one of the most sensitive areas in China to climate changes,but we know little about how current climatic gradients on the plateau influence plant biomass allocation.Gentiana rigescens and G.rhodantha,on the YGP,are important species because they are used in traditional Chinese medicines.We therefore analyzed the biomass allocation patterns of the two species across an elevation gradient(1000–2810 m a.s.l.)on the YGP to understand and predict the impact of climate change on these plant species.We found that the total biomass,reproductive biomass,vegetative biomass,aboveground biomass,and belowground biomass in G.rigescens were all significantly larger than those in G.rhodantha(p<0.05).However,for both species the aboveground biomass was nearly isometrically related to belowground biomass,regardless of elevation,mean annual temperature(MAT)ranging from 8.4℃t to 18.8℃t,and mean annual precipitation(MAP)ranging from 681 to 1327 mm,while the reproductive biomass was allometrically related to vegetative biomass.Intriguingly,there was a significant positive relationship(p<0.05)between the slope of the allometric scaling of reproductive and vegetative biomass and elevation among G.rigescens populations,i.e.plants growing at high elevationsallocate proportionately more biomass to reproduction at larger sizes and less at smaller sizes than plants growing at lower elevations.However,for G.rhodantha the reproductive allocation was negatively correlated with latitude.The results suggested different strategies in reproductive allocation in the two Gentiana plants on the YGP.Further studies are needed to investigate other environmental factors,such as nutrients and light,and genetic factors,in order to understand the trend of reproductive allometry along the environmental gradients.Our study has implications for the management and conservation practices of the two Gentiana species.

Functional characterization of the Arabidopsis SERRATE under salt stress

SERRATE(SE)plays critical roles in RNA metabolism and plant growth regulation.However,its function in stresseresponse processes remains largely unknown.Here,we examined the regulatory role of SE using the se-1 mutant and its complementation line under saline conditions.The expression of SE was repressed by salt treatment at both mRNA and protein levels.After treatment with different NaCl concentrations,the se-1 mutants showed increased sensitivity to salinity.This heightened sensitivity was evidenced by decreased germination,reduced root growth,more serious chlorosis,and increased conductivity of the mutants compared with the wild type.Further analysis revealed that SE regulates the pre-mRNA splicing of several well-characterized marker genes associated with salt stress tolerance.Our data thus imply that SE may function as a key component in plant response to salt stress by modulating the splicing of salt stress-associated genes.

A hybrid sterile locus leads to the linkage drag of interspecific hybrid progenies

Interspecific hybridization plays an important role in rice breeding by broadening access to desirable traits such as disease resistance and improving yields.However,interspecific hybridization is often hindered by hybrid sterility,linkage drag,and distorted segregation.To mine for favorable genes from Oryza glaberrima,we cultivated a series of BC4 introgression lines(ILs)of O.glaberrima in the japonica rice variety background(Dianjingyou 1)in which the IL-2769(BC4F10)showed longer sterile lemmas,wider grains and spreading panicles compared with its receptor parent,suggesting that linkage drag may have occurred.Based on the BC5F2 population,a hybrid sterility locus,S20,a long sterile lemma locus,G1-g,and a new grain width quantitative trait locus(QTL),qGW7,were mapped in the linkage region about 15 centimorgan(cM)from the end of the short arm of chromosome 7.The hybrid sterility locus S20 from O.glaberrima eliminated male gametes of Oryza sativa,and male gametes carrying the alleles of O.sativa in the heterozygotes were aborted completely.In addition,the homozygotes presented a genotype of O.glaberrima,and homozygous O.sativa were not produced.Surprisingly,the linked traits G1-g and qGW7 showed similar segregation distortion.These results indicate that S20 was responsible for the linkage drag.As a large number of detected hybrid sterility loci are widely distributed on rice chromosomes,we suggest that hybrid sterility loci are the critical factors for the linkage drag in interspecific and subspecific hybridization of rice.

Pyrocincholic acid 3β-O-β-D-quinovopyranosyl-28-O-β-Dglucopyranoside suppresses adipogenesis and regulates lipid metabolism in 3T3-L1 adipocytes

Obesity is crucially involved in many metabolic diseases,such as type 2 diabetes,cardiovascular disease and cancer.Regulating the number or size of adipocytes has been suggested to be a potential treatment for obesity.In this study,we investigated the effect of pyrocincholic acid 3β-O-β-D-quinovopyranosyl-28-O-β-D-glucopyranoside(PAQG),a 27-nor-oleanolic acid saponin extracted from Metadina trichotoma,on adipogenesis and lipid metabolism in 3T3-L1 adipocytes.The 3T3-L1 pre-adipocytes were incubated with vehicle or PAQG for 6 days in differentiation process.PAQG significantly reduced the adipogenesis,adiponectin secretion and the expression level of key transcription factors related to adipogenesis,such as PPARc,C/EBPb,C/EBPa,and FABP4.Moreover,PAQG increased the levels of FFA and glycerol in medium and reduced TG level in mature adipocytes.Interestingly,PAQG not only promoted the activation of AMPK and genes involved in fatty oxidation including PDK4 and CPT1a,but also inhibited those genes involved in fatty acid biosynthesis,such as SREBP1c,FAS,ACCa and SCD1.In conclusion,PAQG inhibits the differentiation and regulates lipid metabolism of 3T3-L1 cells via AMPK pathway,suggesting that PAQG may be a novel and promising natural product for the treatment of obesity and hyperlipidemia.

The bHLH Transcription Factors MYC2, MYC3, and MYC4 Are Required for Jasmonate-Mediated Inhibition of Flowering in Arabidopsis

Dear Editor,In plants, the floral transition is flexibly controlled by various environmental conditions and endogenous developmental cues. In Arabidopsis, six major flowering pathways respond to changes in these factors （Fornara et al., 2010）. The photoperiod, vernalization, and ambient pathways monitor exogenous signals from the environment such as day length, minimum winter temperature, and ambient temperature （Fornara et al., 2010）. By contrast, the autonomous, gibberellin, and age pathways respond to endogenous cues linked to developmental status （Fornara et al., 2010）. Accumulating evidence indicates that the six flowering pathways converge in a network to regulate floral integrator genes FLOWERING LOCUS T （FT）, TWIN SISTER OF FT （TSF）, and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 （Fornara et al., 2010）.

Iron uptake, signaling, and sensing in plants

Iron (Fe) is an essential micronutrient that affects the growth and development of plants because it participates as a cofactor in numerous physiological and biochemical reactions. As a transition metal, Fe is redoxactive. Fe often exists in soil in the form of insoluble ferric hydroxides that are not bioavailable to plants.Plants have developed sophisticated mechanisms to ensure an adequate supply of Fe in a fluctuating environment. Plants can sense Fe status and modulate the transcription of Fe uptake-associated genes, finallycontrolling Fe uptake from soil to root. There is a critical need to understand the molecular mechanisms bywhich plants maintain Fe homeostasis in response to Fe fluctuations. This review focuses on recentadvances in elucidating the functions of Fe signaling components. Taking Arabidopsis thaliana and Oryzasativa as examples, this review begins by discussing the Fe acquisition systems that control Fe uptake fromsoil, the major components that regulate Fe uptake systems, and the perception of Fe status. Future explorations of Fe signal transduction will pave the way for understanding the regulatory mechanisms that underlie the maintenance of plant Fe homeostasis.

Natural alleles of a uridine 5'-diphospho-glucosyltransferase gene responsible for differential endosperm development between upland rice and paddy rice

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice. However, the underlying molecular mechanism of this trait is poorly understood. Here,we cloned the uridine 5’-diphospho(UDP)-glucosyltransferase gene EDR1(Endosperm Development in Rice) responsible for differential endosperm development between upland rice and paddy rice by performing quantitative trait loci analysis and map-based cloning. EDR1 was highly expressed in developing seeds duringgrain filling. Natural variations in EDR1 significantly reduced the UDP-glucosyltransferase activity of EDR1 YZNcompared to EDR1 YD1,resulting in abnormal endosperm development in the near-isogenic line, accompanied by insufficient grains and changes in grain quality.By analyzing the distribution of the two alleles EDR1 YD1 and EDR1 YZNamong diverse paddy rice and upland rice varieties, we discovered that EDR1 was conserved in upland rice, but segregated in paddy rice. Further analyses of grain chalkiness in the alleles of EDR1 YD1 and EDR1 YZNvarieties indicated that rice varieties harboring EDR1 YZNand EDR1 YD1 preferentially showed high chalkiness, and low chalkiness,respectively. Taken together, these results suggest that the UDP-glucosyltransferase gene EDR1 is an important determinant controlling differential endosperm development between upland rice and paddy rice.

Biodiversity arks in the Anthropocene

The Anthropocene proposal suggested that the Earth may have entered a new geological epoch,in which human activity and climate change are influencing the environment at global scale.Arrival of the Anthropocene is bringing an unprecedented challenge to the biodiversity that is essential to humans,and enhancing many benefits of nature to human being.However,biodiversity loss is aggravating in the rhythm of inevitable change in the Anthropocene,and the adaptation of biodiversity to the anthropogenic disturbance seems unable to keep pace with the human activity and climate change.Therefore,re-examination of the assumptions and practices upon the current conservation endeavor are needed.We suggested that biodiversity conservation should be paid more attention to the response from biodiversity to the human activity and climate change in the Anthropocene.Thus,the concept of biodiversity arks in the Anthropocene is proposed,that is,biodiversity arks in the Anthropocene are the areas where vulnerable biodiversity is sheltered to alleviate human activity and buffered from climate change under the anthropogenic disturbance.The concept should be implemented for biodiversity conservation to fill gaps between our knowledge and build on successful conservation and sustainability in the Anthropocene.It will be certainly important to conservation policy instruction and management under climate change,especially the implementation of climate buffering zones preserving biodiversity in the face of warming climate.

AtWRKY75 positively regulates age-triggered leaf senescence through gibberellin pathway

WRKY transcription factors play essential roles during leaf senescence.However,the mechanisms by which they regulate this process remains largely unknown.Here,we identified the transcription factor WRKY75 as a positive regulator during leaf senescence.Mutations of WRKY75 caused a delay in agetriggered leaf senescence,whereas overexpression of WRKY75 markedly accelerated this process.Expression of senescence-associated genes(SAGs)was suppressed in WRKY75 mutants but increased in WRKY75-overexpressing plants.Further analysis demonstrated that WRKY75 directly associates with the promoters of SAG12 and SAG29,to activate their expression.Conversely,GAI and RGL1,two DELLA proteins,can suppress the WRKY75-mediated activation,thereby attenuating SAG expression during leaf senescence.Genetic analyses showed that GAI gain-of-function or RGL1 overexpression can partially rescue the accelerated senescence phenotype caused by WRKY75 overexpression.Furthermore,WRKY75 can positively regulate WRKY45 expression during leaf senescence.Our data thus imply that WRKY75 may positively modulate age-triggered leaf senescence through the gibberellin-mediated signaling pathway。

Phytochrome B regulates jasmonic acid-mediated defense response against Botrytis cinerea in Arabidopsis

The phytochrome B mediated light signaling integrates with various phytohormone signalings to control plant immune response.However,it is still unclear whether phyB-mediated light signaling has an effect on the biosynthesis of jasmonate during plant defense response against Botrytis cinerea.In this study,we demonstrated that phyB-mediated light signaling has a role in this process.Initially,we confirmed that phyb plants were obviously less resistant to B.cinerea while phyB overexpressing plants showed significantly enhanced resistance.We also found that the expression of numerous JA biosynthesis genes was promoted upon treatment with red or white light when compared to that of darkness,and that this promotion is dependent on phyB.Consistent with the gene expression results,phyb plants accumulated reduced pool of JA-lle,indicating that phyB-mediated light signaling indeed increased JA biosynthesis.Further genetic analysis showed that light-mediated JAZ9 degradation and phyB-enhanced resistance were dependent on the receptor COI1,and that pifl/3/4/5(pi/q)can largely rescue the severe symptom of phyb.Taken together,our study demonstrates that phyB may participate in plant defense against B.cinerea through the modulation of the biosynthesis of JA.

Arabidopsis WRKY Transcription Factors WRKY12 and WRKY13 Oppositely Regulate Flowering under Short-Day Conditions

In plants, photoperiod is an important cue for determining flowering. The floral transition in Arabidopsis thaliana is earlier under long-day （LD） than under short-day （SD） conditions. Flowering of Arabidopsis plants under SD conditions is mainly regulated by the plant hormone gibberellin （GA）. Here, we report two WRKY transcription factors function oppositely in controlling flowering time under SD conditions. Phenotypic analysis showed that disruption of WRKY12 caused a delay in flowering, while loss of WRKY13 function promoted flowering. WRKY12 and WRKY13 displayed negatively correlated expression profiles and function successively to regulate flowering. Molecular and genetic analyses demonstrated that FRUITFULL （FUL） is a direct downstream target gene of WRKY12 and WRKY13. Interestingly, we found that DELLA proteins GIBBERELLIN INSENSITIVE （GAI） and RGA-LIKE1 （RGL1） interacted with WRKY12 and WRKY13, and their interactions interfered with the transcriptional activity of the WRKY12 and WRKY13. Further studies suggested thatWRKY12 and WRKY13 partly mediated the effect of GA3 on controlling flowering time. Taken together, our results indicate that WRKY12 and WRKY13 oppositely modulate flower- ing time under SD conditions, which at least partially involves the action of GA.

Insight into the mode of action of 2,4-dichlorophenoxyacetic acid(2,4-D) as an herbicide

2,4-Dichlorophenoxyacetic acid （2,4-D） was the first synthetic herbicide to be commercially developed and has commonly been used as a broadleaf herbicide for over 60 years. It is a selective herbicide that kills dicots without affecting monocots and mimics natural auxin at the molecular level. Physiological responses of dicots sensitive to auxinic herbicides include abnormal growth, senescence,and plant death. The identification of auxin receptors, auxin transport carriers, transcription factors response to auxin, and cross-talk among phytohormones have shed light on the molecular action mode of 2,4-D as a herbicide. Here, the molecular action mode of 2,4-D is highlighted according to the latest findings, emphasizing the physiological process, perception, and signal transduction under herbicide treatment.

bHLH121 Functions as a Direct Link that Facilitates the Activation of FIT by bHLH IVc Transcription Factors for Maintaining Fe Homeostasis in Arabidopsis

Iron(Fe)deficiency is prevalent in plants grown in neutral or alkaline soil.Plants have evolved sophisticated mechanisms that regulate Fe homeostasis,ensuring survival.In Arabidopsis,FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR(FIT)is a crucial regulator of Fe-deficiency response.FIT is activated indirectly by basic helix-loop-helix(bHLH)IVc transcription factors(TFs)under Fed eficiency;how ever,it remains unclear which protein(s)act as the linker to mediate the activation of FIT by bHLH IVc TFs.In this study,we characterize the functions of bHLH121 and demonstrate that it directly associates with the FIT promoter.We found that loss-of-function mutations of bHLH121 cause severe Fedeficiency symptoms,reduced Feaccum ulation,and disrupted expression of genes associated with Fehomeostasis.Genetic analysis showed that FIT is epistatic to bHLH121 and FIT overexpression partially rescues the bhlh121 mutant.Further investigations revealed that bHLH IVc TFs interact with and promote nuclear accumulation of bHLH121.We demonstrated that bHLH121 has DNA-binding activity and can bind the prom oters of the FIT and bHLHlb genes,but we did not find that it has either direct transcriptional activation or repression activity tow ard these genes.Meanw hile,we found that bHLH121 functions downstream of and is a direct target of bHLH IVc TFs,and its expression is induced by Fe deficiency in a bHLH IV c-dependent manner.Taken together,these results establish that bHLH121 functions together with bHLH IVc TFs to positively regulate the expression of FIT and thus plays a pivotal role in maintaining Fe homeostasis in Arabidopsis.

Control of sulfate concentration by miR395-targeted APS genes in Arabidopsis thaliana

Sulfur nutrition is crucial for plant growth and development,as well as crop yield and quality.Inorganic sulfate in the soil is the major sulfur source for plants.After uptake,sulfate is activated by ATP sulfurylase,and then gets assimilated into sulfur-containing metabolites.However,the mechanism of regulation of sulfate levels by ATP sulfurylase is unclear.Here,we investigated the control of sulfate levels by miR395-mediated regulation of APS1/3/4.Sulfate was over-accumulated in the shoots of miR395 over-expression plants in which the expression of the APS1,APS3,and APS4 genes was suppressed.Accordingly,reduced expression of miR395 caused a decline of sulfate concentration.In agreement with these results,over-expression of the APS1,APS3,and APS4 genes led to the reduction of sulfate levels.Differential expression of these three APS genes in response to sulfate starvation implied that they have different functions.Further investigation revealed that the regulation of sulfate levels mediated by miR395 depends on the repression of its APS targets.Unlike the APS1,APS3,and APS4 genes,which encode plastid-localized ATP sulfurylases,the APS2 gene encodes a cytosolic version of ATP sulfurylase.Genetic analysis indicated that APS2 has no significant effect on sulfate levels.Our data suggest that miR395-targeted APS genes are key regulators of sulfate concentration in leaves.