Conservation genomic investigation of an endangered conifer,Thuja sutchuenensis,reveals low genetic diversity but also low genetic load

Endangered species generally have small populations with low genetic diversity and a high genetic load.Thuja sutchuenensis is an endangered conifer endemic to southwestern China.It was once considered extinct in the wild,but in 1999 was rediscovered.However,little is known about its genetic load.We collected 67 individuals from five wild,isolated T.sutchuenensis populations,and used 636,151 SNPs to analyze the level of genetic diversity and genetic load in T.sutchuenensis to delineate the conservation units of T.sutchuenensis,based on whole transcriptome sequencing data,as well as target capture sequencing data.We found that populations of T.sutchuenensis could be divided into three groups.These groups had low levels genetic diversity and were moderately genetically differentiated.Our findings also indicate that T.sutchuenensis suffered two severe bottlenecks around the Last Glaciation Period and Last Glacial Maximum.Among Thuja species,T.sutchuenensis presented the lowest genetic load and hence might have purged deleterious mutations efficiently through purifying selection.However,distribution of fitness effects analysis indicated a high extinction risk for T.sutchuenensis.Multiple lines of evidence identified three management units for T.sutchuenensis.Although T.sutchuenensis possesses a low genetic load,low genetic diversity,suboptimal fitness,and anthropogenic pressures all present an extinction risk for this rare conifer.This might also hold true for many endangered plant species in the mountains all over the world.

Across two phylogeographic breaks: Quaternary evolutionary history of a mountain aspen (Populus rotundifolia) in the Hengduan Mountains

Biogeographical barriers to gene flow are central to plant phylogeography.In East Asia,plant distribution is greatly influenced by two phylogeographic breaks,the Mekong-Salween Divide and Tanaka-Kaiyong Line,however,few studies have investigated how these barriers affect the genetic diversity of species that are distributed across both.Here we used 14 microsatellite loci and four chloroplast DNA fragments to examine genetic diversity and distribution patterns of 49 populations of Populus rotundifolia,a species that spans both the Mekong-Salween Divide and the Tanaka-Kaiyong Line in southwestern China.Demographic and migration hypotheses were tested using coalescent-based approaches.Limited historical gene flow was observed between the western and eastern groups of P.rotundifolia,but substantial flow occurred across both the Mekong-Salween Divide and Tanaka-Kaiyong Line,manifesting in clear admixture and high genetic diversity in the central group.Wind-borne pollen and seeds may have facilitated the dispersal of P.rotundifolia following prevalent northwest winds in the spring.We also found that the Hengduan Mountains,where multiple genetic barriers were detected,acted on the whole as a barrier between the western and eastern groups of P.rotundifolia.Ecological niche modeling suggested that P.rotundifolia has undergone range expansion since the last glacial maximum,and demographic reconstruction indicated an earlier population expansion around 600 Ka.The phylogeographic pattern of P.rotundifolia reflects the interplay of biological traits,wind patterns,barriers,niche differentiation,and Quaternary climate history.This study emphasizes the need for multiple lines of evidence in understanding the Quaternary evolution of plants in topographically complex areas.

Effects of taxon sampling on molecular dating for within-genus divergence events,when deep fossils are used for calibration

A universal method of molecular dating that can be applied to all families and genera regardless of their fossil records, or lack thereof, is highly desirable. A possible method for eudicots is to use a large phylogeny calibrated using deep fossils including tricolpate pollen as a fixed （124 mya） calibration point. This method was used to calculate node ages within three species-poor disjunct basal eudicot genera, Caulophyllum, Podophyllum and Pachysandra, and sensitivity of these ages to effects such as taxon sampling were then quantified. By deleting from one to three accessions related to each genus in 112 different combinations, a confidence range describing variation due only to taxon sampling was generated. Ranges for Caulophyllum, Podophyllum and Pachysandra were 8.4-10.6, 7.6-20.0, and 17.6-25.0 mya, respectively. However, the confidence ranges calculated using bootstrapping were much wider, at 3-19, 0-32 and 11-32 mya, respectively. Furthermore, deleting 10 adjacent taxa had a large effect in Pachysandra only, indicating that undersampling effects are significant among Buxales. Changes to sampling density in neighboring clades, or to the position of the fixed fossil calibration point had small to negligible effects. Non-parametric rate smoothing was more sensitive to taxon sampling effects than was penalized likelihood. The wide range for Podophyllum, compared to the other two genera, was probably due to a high degree of rate heterogeneity within this genus. Confidence ranges calculated by this method could be narrowed by sampling more individuals within the genus of interest, and by sequencing multiple DNA regions from all species in the phylogeny.

Boehmeria nivea var. strigosa (Urticaceae), a new variety from Southwest China

Boehmeria nivea var.strigosa Zeng Y.Wu&Y.Zhao,a new variety of B.nivea(Urticaceae)from Southwest China,is here described based on evidence from morphology and molecular phylogeny.This new variety is mainly characterized by its green abaxial leaf blade,partly connate stipules,and densely patent strigose hairs on stems and potioles.The phylogenetic analysis based on rbc L,nrDNA and rbc L+nrDNA datasets,revealed that all individuals of B.nivea var.strigosa formed a monophyletic group.The conservation status of B.nivea var.strigosa is assessed as“Near Threatened”(NT)according to IUCN evaluation criteria.The discovery of this new variety is not only crucial for the taxonomy of ramie,but also provides reference for the exploration and utilization of ramie.

Genomic divergence and mutation load in the Begonia masoniana complex from limestone karsts

Understanding genome-wide diversity,inbreeding,and the burden of accumulated deleterious mutations in small and isolated populations is essential for predicting and enhancing population persistence and resilience.However,these effects are rarely studied in limestone karst plants.Here,we re-sequenced the nuclear genomes of 62 individuals of the Begonia masoniana complex(B.liuyanii,B.longgangensis,B.masoniana and B.variegata)and investigated genomic divergence and genetic load for these four species.Our analyses revealed four distinct clusters corresponding to each species within the complex.Notably,there was only limited admixture between B.liuyanii and B.longgangensis occurring in overlapping geographic regions.All species experienced historical bottlenecks during the Pleistocene,which were likely caused by glacial climate fluctuations.We detected an asymmetric historical gene flow between group pairs within this timeframe,highlighting a distinctive pattern of interspecific divergence attributable to karst geographic isolation.We found that isolated populations of B.masoniana have limited gene flow,the smallest recent population size,the highest inbreeding coefficients,and the greatest accumulation of recessive deleterious mutations.These findings underscore the urgency to prioritize conservation efforts for these isolated population.This study is among the first to disentangle the genetic differentiation and specific demographic history of karst Begonia plants at the whole-genome level,shedding light on the potential risks associated with the accumulation of deleterious mutations over generations of inbreeding.Moreover,our findings may facilitate conservation planning by providing critical baseline genetic data and a better understanding of the historical events that have shaped current population structure of rare and endangered karst plants.

Genetic analysis of walnut cultivars from southwest China:Implications for germplasm improvement

Walnuts are highly valued for their rich nutritional profile and wide medicinal applications.This demand has led to the intensification of breeding activities in major walnut production areas such as southwest China,in order to develop more superior cultivars.With the increasing number of cultivars,accurate identification becomes fundamental to selecting the right cultivar for grafting,industrial processing or development of new cultivars.To ensure proper identification of cultivars and understand the genetic structure of wild and cultivated material,we genotyped 362 cultivated and wild individuals of walnut trees from southwest China(with two additional populations from Xinjiang,plus three cultivars from Canada,France and Belgium) using 36 polymorphic microsatellite loci.We found relatively low indices of genetic diversity(H_(O)=0.570,H_(E)=0.404,N_(A)=2.345) as well as a high level of clonality(>85% of cultivars),indicating reliance on genetically narrow sources of parental material for breeding.Our STRUCTURE and PCoA analyses generally delineated the two species,though considerable levels of introgression were also evident.More significantly,we detected a distinct genetic group of cultivated Juglans sigillata,which mainly comprised individuals of the popular ’Yangbidapao’ landrace.Finally,a core set of 18 SSR loci was selected,which was capable of identifying 32 cultivars.In a nutshell,our results call for more utilization of genetically disparate material,including wild walnut trees,as parental sources to breed for more cultivars.The data reported herein will significantly contribute towards the genetic improvement and conservation of the walnut germplasm in southwest China.

Nitric oxide regulation of plant metabolism

Nitric oxide(NO)has emerged as an important signal molecule in plants,having myriad roles in plant devel-opment.In addition,NO also orchestrates both biotic and abiotic stress responses,during which intensive cellular metabolic reprogramming occurs.Integral to these responses is the location of NO biosynthetic and scavenging pathways in diverse cellular compartments,enabling plants to effectively organize signal transduction pathways.NO regulates plant metabolism and,in turn,metabolic pathways reciprocally regu-late NO accumulation and function.Thus,these diverse cellular processes are inextricably linked.This re-view addresses the numerous redox pathways,located in the various subcellular compartments that pro-duce NO,in addition to the mechanisms underpinning NO scavenging.We focus on how this molecular dance is integrated into the metabolic state of the cell.Within this context,a reciprocal relationship be-tween NO accumulation and metabolite production is often apparent.We also showcase cellular pathways,including those associated with nitrate reduction,that provide evidence for this integration of NO function and metabolism.Finally,we discuss the potential importance of the biochemical reactions governing NO levels in determining plant responses to a changing environment.

Hetero-trans-β-Glucanase Produces Cellulose-Xyloglucan Covalent Bonds in the Cell Walls of Structural Plant Tissues and Is Stimulated by Expansin

Current cell-wall models assume no covalent bonding between cellulose and hemicelluloses such as xyloglu-can or mixed-linkageβ-D-glucan(MLG).However,Equisetum hetero-trans-β-glucanase(HTG)grafts cellu-lose onto xyloglucan oligosaccharides(XGOs)-and,we now show,xyloglucan polysaccharide-in vitro,thus exhibiting CXE(cellulose:xyloglucan endotransglucosylase)activity.In addition,HTG also catalyzes MLG-to-XGO bonding(MXE activity).In this study,we explored the CXE action of HTG in native plant cell walls and tested whether expansin exposes cellulose to HTG by disrupting hydrogen bonds.To quantify and visu-alize CXE and MXE action,we assayed the sequential release of HTG products from cell walls pre-labeled with substrate mimics.We demonstrated covalent cellulose--xyloglucan bonding in plant cell walls and showed that CXE and MXE action was up to 15%and 60%of total transglucanase action,respectively,and peaked in aging,strengthening tissues:CXE in xylem and cells bordering intercellular canals and MXE in scleren-chyma.Recombinant bacterial expansin(EXLX1)strongly augmented CXE activity in vitro.CXE and MXE ac-tion in living Equisetum structural tissues potentially strengthens stems,while expansin might augment the HTG-catalyzed CXE reaction,thereby allowing efficient CXE action in muro.Our methods will enable surveys for comparable reactions throughout the plant kingdom.Furthermore,engineering similar hetero-polymer formation into angiosperm crop plants may improve certain agronomic traits such as lodging tolerance.

An activated form of NB-ARC protein RLS1 functions with cysteine-rich receptor-like protein RMC to trigger cell death in rice

A key event that follows pathogen recognition by a resistance(R)protein containing an NB-ARC(nucleotide-binding adaptor shared by Apaf-1,R proteins,and Ced-4)domain is hypersensitive response(HR)-type cell death accompanied by accumulation of reactive oxygen species and nitric oxide.However,the integral mechanisms that underlie this process remain relatively opaque.Here,we show that a gain-offunction mutation in the NB-ARC protein RLS1(Rapid Leaf Senescence 1)triggers high-light-dependent HR-like cell death in rice.The RLS1-mediated defense response is largely independent of salicylic acid accumulation,NPR1(Nonexpressor of Pathogenesis-Related Gene 1)activity,and RAR1(Required for Mla12 Resistance 1)function.A screen for suppressors of RLS1 activation identified RMC(Root Meander Curling)as essential for the RLS1-activated defense response.RMC encodes a cysteine-rich receptor-like secreted protein(CRRSP)and functions as an RLS1-binding partner.Intriguingly,their co-expression resulted in a change in the pattern of subcellular localization and was sufficient to trigger cell death accompanied by a decrease in the activity of the antioxidant enzyme APX1.Collectively,our findings reveal an NBARC-CRRSP signaling module that modulates oxidative state,the cell death process,and associated immunity responses in rice.

Phenolic metabolism and molecular mass distribution of polysaccharides in cellulose-deficient maize cells

As a consequence of the habituation to low levels of dichlobenil （DCB）, cultured maize cells presented an altered hemicellulose cell fate with a lower proportion of strongly wall-bound hemicelluloses and an increase in soluble extracellular polymers released into the culture medium. The aim of this study was to investigate the relative molecular mass distributions of polysaccharides as well as phenolic metabolism in cells habituated to low levels of DCB （1.5 μM）. Generally, cell wall bound hemicelluloses and sloughed polymers from habituated cells were more homogeneously sized and had a lower weight-average relative molecular mass. In addition, polysaccharides underwent massive cross-linking after being secreted into the cell wall, but this cross-linking was less pronounced in habituated cells than in non-habituated ones. However, when relativized, ferulic acid and p-coumaric acid contents were higher in this habituated cell line. Feasibly, cells habituated to low levels of DCB synthesized molecules with a lower weight-average relative molecular mass, although cross-linked, as a part of their strategy to compensate for the lack of cellulose.

New insights into nitric oxide biosynthesis underpin lateral root development

In recent years,nitric oxide(NO)has emerged as a key redox signaling molecule in plants functioning in the regulation of key developmental programs and the orchestration of responses to a plethora of environmental cues(Kolbert et al.,2019).The predominant route for the transfer of NO bioactivity is through S-nitrosylation,the addition of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol(Yun et al.,2011).Specificity for this process is established by the structural constraints imposed by tertiary protein structure in gating access to given cysteine redox switches and associated proteins that can either facilitate the addition or removal of the NO moiety at these residues(Umbreen et al.,2018).

Genomic variation,environmental adaptation,and feralization in ramie,an ancient fiber crop

Feralization is an important evolutionary process,but the mechanisms behind it remain poorly understood.Here,we use the ancient fiber crop ramie(Boehmeria nivea(L.)Gaudich.)as a model to investigate genomic changes associated with both domestication and feralization.We first produced a chromosome-scale de novo genome assembly of feral ramie and investigated structural variations between feral and domesticated ramie genomes.Next,we gathered 915 accessions from 23 countries,comprising cultivars,major landraces,feral populations,and the wild progenitor.Based on whole-genome resequencing of these accessions,we constructed the most comprehensive ramie genomic variation map to date.Phylogenetic,demographic,and admixture signal detection analyses indicated that feral ramie is of exoferal or exo-endo origin,i.e.,descended from hybridization between domesticated ramie and the wild progenitor or ancient landraces.Feral ramie has higher genetic diversity than wild or domesticated ramie,and genomic regions affected by natural selection during feralization differ from those under selection during domestication.Ecological analyses showed that feral and domesticated ramie have similar ecological niches that differ substantially from the niche of the wild progenitor,and three environmental variables are associated with habitat-specific adaptation in feral ramie.These findings advance our understanding of feralization,providing a scientific basis for the excavation of new crop germplasm resources and offering novel insights into the evolution of feralization in nature.

H_2O_2-induced Leaf Cell Death and the Crosstalk of Reactive Nitric/Oxygen Species

In plants, the chloroplast is the main reactive oxygen species （ROS） producing site under high light stress. Catalase （CAT）, which decomposes hydrogen peroxide （H2O2）, is one of the controlling enzymes that maintains leaf redox homeostasis. The catalase mutants with reduced leaf catalase activity from different plant species exhibit an H2O2-induced leaf cell death phenotype. This phenotype was differently affected by light intensity or photoperiod, which may be caused by plant species, leaf redox status or growth conditions. In the rice CAT mutant nitric oxide excess 1 （noe1）, higher H2O2 levels induced the generation of nitric oxide （NO） and higher S-nitrosothiol （SNO） levels, suggesting that NO acts as an important endogenous mediator in H2O2-induced leaf cell death. As a free radical, NO could also react with other intracellular and extracellular targets and form a series of related molecules, collectively called reactive nitrogen species （RNS）. Recent studies have revealed that both RNS and ROS are important partners in plant leaf cell death. Here, we summarize the recent progress on H2O2-induced leaf cell death and the crosstalk of RNS and ROS signals in the plant hypersensitive response （HR）, leaf senescence, and other forms of leaf cell death triggered by diverse environmental conditions.

The many roles of small RNAs in leaf development

Leaf development involves many complex genetic interactions,signals between adjacent cells or between more distant tissues and consequent changes in cell fate.This review describes three stages in leaf development where regulation by small RNAs have been used to modulate gene expression patterns.

Changes in Cinnamic Acid Derivatives Associated with the Habituation of Maize Cells to Dichlobenil

The habituation of cell cultures to cellulose biosynthesis inhibitors such as dichlobenil （DCB） represents a valu- able tool to improve our knowledge of the mechanisms involved in plant cell wall structural plasticity. Maize cell lines habituated to lethal concentrations of DCB were able to grow through the acquisition of a modified cell wall in which cellulose was partially replaced by a more extensive network of arabinoxylans. The aim of this work was to investigate the phenolic metabolism of non-habituated and DCB-habituated maize cell cultures. Maize cell cultures were fed [14C]cinnamate and the fate of the radioactivity in different intra-protoplasmic and wall-localized fractions throughout the culture cycle was analyzed by autoradiography and scintillation counting. Non-habituated and habituated cultures did not markedly differ in their ability to uptake exogenous [14C]cinnamic acid. However, interesting differences were found in the radiolabeling of low- and high-Mr metabolites. Habituated cultures displayed a higher number and amount of radiola-beled low-Mr compounds, which could act as reserves later used for polysaccharide feruloylation. DCB-habituated cultures were highly enriched in esterified [14C]dehydrodiferulates and larger coupling products. In conclusion, an extensive and early cross-linking of hydroxycinnamates was observed in DCB-habituated cultures, probably strengthening their cellulose-deficient walls.

Nitric oxide: promoter or suppressor of programmed cell death?

Nitric oxide(NO)is a short-lived gaseous free radical that predominantly functions as a messenger and effector molecule.It affects a variety of physiological processes,including programmed cell death(PCD)through cyclic guanosine monophosphate(cGMP)-dependent and-independent pathways.In this field,dominant discoveries are the diverse apoptosis networks in mammalian cells,which involve signals primarily via death receptors(extrinsic pathway)or the mitochondria(intrinsic pathway)that recruit caspases as effector molecules.In plants,PCD shares some similarities with animal cells,but NO is involved in PCD induction via interacting with pathways of phytohormones.NO has both promoting and suppressing effects on cell death,depending on a variety of factors,such as cell type,cellular redox status,and the flux and dose of local NO.In this article,we focus on how NO regulates the apoptotic signal cascade through protein S-nitrosylation and review the recent progress on mechanisms of PCD in both mammalian and plant cells.

The biosynthesis and wall-binding of hemicelluloses in cellulose-deficient maize cells:An example of metabolic plasticity

Cell-suspension cultures（Zea mays L.,Black Mexican sweet corn） habituated to 2,6-dichlorobenzonitrile（DCB） survive with reduced cellulose owing to hemicellulose network modification. We aimed to de fine the hemicellulose metabolism modifications in DCB-habituated maize cells showing a mild reduction in cellulose at different stages in the culture cycle. Using pulse-chase radiolabeling, we fed habituated and non-habituated cultures with [3H]arabinose,and traced the distribution of 3H-pentose residues between xylans, xyloglucans and other polymers in several cellular compartments for 5 h. Habituated cells were slower taking up exogenous [3H]arabinose. Tritium was incorporated into polysaccharide-bound arabinose and xylose residues, but habituated cells diverted a higher proportion of their new [3H]xylose residues into（hetero） xylans at the expense of xyloglucan synthesis. During logarithmic growth, habituated cells showed slower vesicular traf ficking of polymers,especially xylans. Moreover, habituated cells showed a decrease in the strong wall-binding of all pentose-containing polysaccharides studied; correspondingly, especially in log phase cultures, habituation increased the proportion of 3H-hemicelluloses（[3H]xylans and [3H]xyloglucan） sloughed into the medium. These findings could be related to the cel walls’ cellulose-deficiency, and consequent reduction in binding sites for hemicelluloses; the data could also re fl ect the habituated cells’ reduced capacity to integrate arabinox ylans by extra-protoplasmic phenolic cross-linking, as well as xyloglucans, during wall assembly.

A general method for assaying homo-and hetero-transglycanase activities that act on plant cell-wall polysaccharides

Transglycanases（endotransglycosylases） cleave a polysaccharide（donor-substrate） in mid-chain, and then transfer a portion onto another poly-or oligosaccharide（acceptor-substrate）. Such enzymes contribute to plant cellwall assembly and/or re-structuring. We sought a general method for revealing novel homo- and hetero-transglycanases, applicable to diverse polysaccharides and oligosaccharides, separating transglycanase-generated3 Hpolysaccharides from unreacted3H-oligosaccharides—the former immobilized（on filter-paper, silica-gel or glassfiber）,the latter eluted. On filter-paper, certain polysaccharides [e.g.（1!3, 1!4）-b-D-glucans] remained satisfactorily adsorbed when water-washed; others（e.g. pectins） were partially lost. Many oligosaccharides（e.g. arabinan-, galactan-, xyloglucan-based） were successfully eluted in appropriate solvents, but others（e.g. [3H]xylohexaitol, [3H]mannohexaitol[3H]cellohexaitol） remained immobile. On silica-gel, all3 Holigosaccharides left an immobile ‘ghost’ spot（contaminating any3H-polysaccharides）, which was diminished but not prevented by additives e.g. sucrose or Triton X-100. The best stratum was glassfiber（GF）, onto which the reactionmixture was dried then washed in 75% ethanol. Washing led to minimal loss or lateral migration of3H-polysaccharides if conducted by slow percolation of acidified ethanol. The effectiveness of GF-blotting was well demonstrated for Chara vulgaris transb-mannanase. In conclusion, our novel GF-blotting technique ef ficiently frees transglycanase-generated3H-polysaccharides from unreacted3H-oligosaccharides,enabling high-throughput screening of multiple postulated transglycanase activities utilising chemically diverse donorand acceptor-substrates.

Plant Biosystems Design Research Roadmap 1.0

Human life intimately depends on plants for food,biomaterials,health,energy,and a sustainable environment.Various plants have been genetically improved mostly through breeding,along with limited modification via genetic engineering,yet they are still not able to meet the ever-increasing needs,in terms of both quantity and quality,resulting from the rapid increase in world population and expected standards of living.A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches.This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems.Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes.From this perspective,we present a comprehensive roadmap of plant biosystems design covering theories,principles,and technical methods,along with potential applications in basic and applied plant biology research.We highlight current challenges,future opportunities,and research priorities,along with a framework for international collaboration,towards rapid advancement of this emerging interdisciplinary area of research.Finally,we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception,trust,and acceptance.

Profile of Anthony Trewavas

When asked to write on my experience in science and give some background, it sounded remarkably easy at first. In fact, it has proved very difficult. Apart from the short introduction below, I decided to write on four topics to which I have made a minor contribution. These are used to illustrate how I often fumbled my way through research and the ideas that I found fascinating. Investigation is never a direct path but a road full of obstacles.