BRITTLE CULM16(BRITTLE NODE) is required for the formation of secondary cell walls in rice nodes

Plant cell walls constitute the skeletal structures of plant bodies,and thus confer lodging resistance for grain crops.While the basic cell wall synthesis machinery is relatively well established now,our understanding of how the process is regulated remains limited and fragmented.In this study,we report the identification and characterization of the novel rice（Oryza sativa L.）brittle culm16（brittle node;bc16）mutant.The brittle node phenotype of the bc16 mutant appears exclusively at nodes,and resembles the previously reported bc5 mutant.Combined histochemical staining and electron microscopy assays revealed that in the bc16 mutant,the secondary cell wall formation and thickening of node sclerenchyma tissues are seriously affected after heading.Furthermore,cell wall composition assays revealed that the bc16 mutation led to a significant reduction in cellulose and lignin contents.Using a map-based cloning approach,the bc16 locus is mapped to an approximately 1.7-Mb region of chromosome 4.Together,our findings strengthen evidence for discretely spatial differences in the secondary cell wall formation within plant bodies.

Defining the Transition from Cell Elongation to Secondary Cell Wall Biosynthesis:Promoter Analyses,Transcript Profiling,and Genomic Analysis of Near-isogenic Germplasms that Differ in Fiber Strength

A distinct set of homoeologous cellulose synthase catalytic subunit(CesA) genes are coordinately up-regulated with the onset of secondary wall formation in cotton fiber as shown by quantitative-RT-

Strigolactones modulate cotton fiber elongation and secondary cell wall thickening

Cotton is one of the most important economic crops in the world,and it is a major source of fiber in the textile industry.Strigolactones(SLs)are a class of carotenoid-derived plant hormones involved in many processes of plant growth and development,although the functions of SL in fiber development remain largely unknown.Here,we found that the endogenous SLs were significantly higher in fibers at 20 days post-anthesis(DPA).Exogenous SLs significantly increased fiber length and cell wall thickness.Furthermore,we cloned three key SL biosynthetic genes,namely GhD27,GhMAX3,and GhMAX4,which were highly expressed in fibers,and subcellular localization analyses revealed that GhD27,GhMAX3,and GhMAX4 were localized in the chloroplast.The exogenous expression of GhD27,GhMAX3,and GhMAX4 complemented the physiological phenotypes of d27,max3,and max4 mutations in Arabidopsis,respectively.Knockdown of GhD27,GhMAX3,and GhMAX4 in cotton resulted in increased numbers of axillary buds and leaves,reduced fiber length,and significantly reduced fiber thickness.These findings revealed that SLs participate in plant growth,fiber elongation,and secondary cell wall formation in cotton.These results provide new and effective genetic resources for improving cotton fiber yield and plant architecture.

The RLCK-VND6 module coordinates secondary cell wall formation and adaptive growth in rice

The orderly deposition of secondary cell wall(SCW)in plants is implicated in various biological programs and is precisely controlled.Although many positive and negative regulators of SCW have been documented,the molecular mechanisms underlying SCW formation coordinated with distinct cellular physiological processes during plant adaptive growth remain largely unclear.Here,we report the identification of Cellulose Synthase co-expressed Kinase1(CSK1),which encodes a receptor-like cytoplasmic kinase,as a negative regulator of SCW formation and its signaling cascade in rice.Transcriptome deep sequencing of developing internodes and genome-wide co-expression assays revealed that CSK1 is co-expressed with cellulose synthase genes and is responsive to various stress stimuli.The increased SCW thickness and vigorous vessel transport in csk1 indicate that CSK1 functions as a negative regulator of SCW biosynthesis.Through observation of green fluorescent protein-tagged CSK1 in rice protoplasts and stable transgenic plants,we found that CSK1 is localized in the nucleus and cytoplasm adjacent to the plasma membrane.Biochemical and molecular assays demonstrated that CSK1 phosphorylates VASCULAR-RELATED NAC-DOMAIN 6(VND6),a master SCW-associated transcription factor,in the nucleus,which reduces the transcription of a suite of SCW-related genes,thereby attenuating SCW accumulation.Consistently,genetic analyses show that CSK1 functions upstream of VND6 in regulating SCW formation.Interestingly,our physiological analyses revealed that CSK1 and VND6 are involved in abscisic acid-mediated regulation of cell growth and SCW deposition.Taken together,these results indicate that the CSK1-VND6 module is an important component of the SCW biosynthesis machinery,which coordinates SCW accumulation and adaptive growth in rice.Our study not only identifies a new regulator of SCW biosynthesis but also reveals a fine-tuned mechanism for precise control of SCW deposition,offering tools for rationally tailoring agronomic traits.

On-Off Switches for Secondary Cell Wall Biosynthesis

Secondary cell walls provide plants with rigidity and strength to support their body weight and ensure water and nutrient transport. They also provide textiles, timber, and potentially second-generation biofuels for human use. Genes responsible for synthesis of the different cell wall components, namely cellulose, hemicelluloses, and lignin, are coordinately expressed and under transcriptional regulation. In the past several years, cell wall-related NAC and MYB transcription factors have been intensively investigated in different species and shown to be master switches of secondary cell wall biosynthesis. Positive and negative regulators, which function upstream of NAC master switches, have also been identified in different plant tissues. Further elucidation of the regulatory mechanisms of cell wall synthesis will facilitate the engineering of plant feedstocks suitable for biofuel production.

ZmMs33 promotes anther elongation via modulating cell elongation regulators,metabolic homeostasis,and cell wall remodeling in maize

Plant cell elongation depends on well-defined gene regulations,adequate nutrients,and timely cell wall modifications.Anther size is positively correlated with the number and viability of pollen grains,while little is known about molecular mechanisms underlying anther cell elongation.Here,we found that properly activated cell elongation regulators at transcriptional levels in loss-of-function ZmMs33 mutant(ms33-6038)anthers failed to promote maize anther elongation.ZmMs33 deficiency disrupted metabolic homeostasis mainly by inhibiting both photosynthesis in anther endothecium and lipid accumulation in anther tapetum.Importantly,ms33-6038 anthers displayed ectopic,premature and excessive secondary cell wall thickening in anther middle layer,which constrained cell elongation structurally and blocked nutrient flows across different anther wall layers.The metabolic disorder was only found in ms33-6038 mutant rather than several representative male-sterility lines at transcriptional and post-translational levels.Collectively,the disordered metabolisms and blocked nutrient flows defeated the activated cell elongation regulators,and finally inhibited anther elongation and growth with a unique‘‘idling effect”in ms33-6038 mutant.

Secondary Cell Wall Deposition in Developing Secondary Xylem of Poplar

Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing secondary xylem cells from hybrid poplar （Populus deltoides x P. trichocarpa）, which were actively making secondary cell walls, were preserved with high pressure freezing/freeze substitution for light and electron microscopy. The distribution of xylans and mannans in the different cell types of developing secondary xylem were detected with immunofluorescence and immuno-gold labeling. While xylans, detected with the monoclonal antibody LM10, had a general distribution across the secondary xylem, mannans were enriched in the S2 secondary cell wall layer of fibers. To observe the cellular structures associated with secondary wall production, cryofixed fibers were examined with transmission electron microscopy during differentiation. There were abundant cortical microtubules and endomembrane activity in cells during the intense phase of secondary cell wall synthesis. Microtubuleassociated small membrane compartments were commonly observed, as well as Golgi and secretory vesicles fusing with the plasma membrane.

GhKNL1 controls fiber elongation and secondary cell wall synthesis by repressing its downstream genes in cotton(Gossypium hirsutum)

Cotton which produces natural fiber materials for the textile industry is one of the most important crops in the world. Class II KNOX proteins are often considered as transcription factors in regulating plant secondary cell wall(SCW) formation. However,the molecular mechanism of the KNOX transcription factor-regulated SCW synthesis in plants(especially in cotton) remains unclear in details so far. In this study, we show a cotton class II KNOX protein(Gh KNL1) as a transcription repressor functioning in fiber development. The Gh KNL1-silenced transgeniccotton produced longer fibers with thicker SCWs,whereas Gh KNL1 dominant repression transgenic lines displayed the opposite fiber phenotype, compared with controls. Further experiments revealed that Gh KNL1 could directly bind to promoters of Gh Ces A4-2/4-4/8-2 and Gh MYB46 for modulating cellulose synthesis during fiber SCW development in cotton. On the other hand, Gh KNL1 could also suppress expressions of Gh EXPA2 D/4 A-1/4 D-1/13 A through binding to their promoters for regulating fiber elongation of cotton. Taken together, these data revealed Gh KNL1 functions in fiber elongation and SCW formation by directly repressing expressions of its target genes related to cell elongation and cellulose synthesis. Thus, our data provide an effective clue for potentially improving fiber quality by genetic manipulation of Gh KNL1 in cotton breeding.

A Phytochrome B-PIF4-MYC2/MYC4 module inhibits secondary cell wall thickening in response to shaded light

Secondary cell walls(SCWs)in stem cells provide mechanical strength and structural support for growth.SCW thickening varies under different light conditions.Our previous study revealed that blue light enhances SCW thickening through the redundant function of MYC2 and MYC4 directed by CRYPTOCHROME1(CRY1)signaling in fiber cells of the Arabidopsis inflorescence stem.In this study,we find that the Arabidopsis PHYTOCHROME B mutant phyB displays thinner SCWs in stem fibers,but thicker SCWs are deposited in the PHYTOCHROME INTERACTING FACTOR(PIF)quadruple mutant pif1pif3pif4pif5(pifq).The shaded light condition with a low ratio of red to far-red light inhibits stem SCW thickening.PIF4 interacts with MYC2 and MYC4 to affect their localization in nuclei,and this interaction results in inhibition of the MYCs’transactivation activity on the NST1 promoter.Genetic evidence shows that regulation of SCW thickening by PIFs is dependent on MYC2/MYC4 function.Together,the results of this study reveal a PHYB-PIF4-MYC2/MYC4 module that inhibits SCW thickening in fiber cells of the Arabidopsis stem.

Isolation and characterization of the secondary wall-related SND1 gene in hawthorn

Secondary wall-associated NAC domain protein1 （SND1） is a key regulator directly regulating the expression levels of MYB46 and MYB83 in the regulation network for secondary wall synthesis, especially in plant fibres. In this study, a SND1 gene was isolated from hawthorn （Crataegus pinnatifida） and named as CpSND 1 because it has a conservative N-terminal DNA- binding domain with AtSNDI. Arabidopsis plants overexpressing CpSND1 had similar phenotypes as plants overexpressing AtSND1, including inhibited growth, upward-curling leaves, sepal dysplasia and sterility. In addition, overexpressing CpSNDI in Arabidopsis also induced the expression of downstream genes, including lignin, cellulose and xylan biosynthesis genes as well as MYB genes. Our results provided functional information of CpSND1 for future genetic engineering in hawthorn.

Combining single-cell RNA sequencing with spatial transcriptome analysis reveals dynamic molecular maps of cambium differentiation in the primary and secondary growth of trees

Primary and secondary growth of the tree stem are responsible for corresponding increases in trunk height and diameter.However,our molecular understanding of the biological processes that underlie these two types of growth is incomplete.In this study,we used single-cell RNA sequencing and spatial transcriptome sequencing to reveal the transcriptional landscapes of primary and secondary growth tissues in the Populus stem.Comparison between the cell atlas and differentiation trajectory of primary and secondary growth revealed different regulatory networks involved in cell differentiation from cambium to xylem precursors and phloem precursors.These regulatory networks may be controlled by auxin accumulation and distribution.Analysis of cell differentiation trajectories suggested that vessel and fiber development followed a sequential pattern of progressive transcriptional regulation.This research provides new insights into the processes of cell identity and differentiation that occur throughout primary and secondary growth of tree stems,increasing our understanding of the cellular differentiation dynamics that occur during stemgrowth in trees.

Disruption of Secondary Wall Cellulose Biosynthesis Alters Cadmium Translocation and Tolerance in Rice Plants

Tricheary elements （TEs）, wrapped by secondary cell wall, play essential roles in water, mineral, and nutrient transduction. Cadmium （Cd） is a toxic heavy metal that is absorbed by roots and transported to shoot, leaves, and grains through vascular systems in plants. As rice is a major source of Cd intake, many efforts have been made to establish ＇low- Cd rice＇. However, no links have been found between cellulose biosynthesis and cadmium accumulation. We report here a rice brittle culm13 mutant, resulting from a novel missense mutation （G101K） in the N-terminus of cellulose synthase subunit 9 （CESA9）. Except for the abnormal mechanical strength, the mutant plants are morphologically indistinguishable from the wild-type plants. Transmission electron microscopy （TEM） and chemical analyses showed a slight reduction in secondary wall thickness and 22% decrease in cellulose content in bc13 plants. Moreover, this mutation unexpectedly confers the mutant plants Cd tolerance due to less Cd accumulation in leaves. Expression analysis of the genes required for Cd uptake and transport revealed complicated alterations after applying Cd to wild-type and bc13. The mutants were further found to have altered vascular structure. More importantly, Cd concentration in the xylem saps from the bc13 plants was significantly lower than that from the wild-type. Combining the analyses of CESA9 gene expression and Cd content retention in the cell-wall residues, we conclude that CESA9^G101K mutation alters cell-wall properties in the conducting tissues, which consequently affects Cd translocation efficiency that largely contributes to the low Cd accumulation in the mutant plants.

Global Analysis of Direct Targets of Secondary Wall NAC Master Switches in Arabidopsis

We report the genome-wide analysis of direct target genes of SND1 and VND7, two Arabidopsis thaliana NAC domain transcription factors that are master regulators of secondary wall biosynthesis in fibers and vessels, respectively. Systematic mapping of the SND1 binding sequence using electrophoretic mobility shift assay and transactivation analysis demonstrated that SND1 together with other secondary wall NACs （SWNs）, including VND6, VND7, NST1, and NST2, bind to an imperfect palindromic 19-bp consensus sequence designated as secondary wall NAC binding element （SNBE）, （T/A）NN（C/T） （TICIG）TNNNNNNNA（AIC）GN（AJCIT） （A/T）, in the promoters of their direct targets. Genome-wide analysis of direct targets of SND1 and VND7 revealed that they directly activate the expression of not only downstream transcription factors, but also a number of non-transcription factor genes involved in secondary wall biosynthesis, cell wall modification, and programmed cell death, the promoters of which all contain multiple SNBE sites. SND1 and VND7 directly regulate the expression of a set of common targets but each of them also preferentially induces a distinct set of direct targets, which is likely attributed to their differential activation strength toward SNBE sites. Complementation study showed that the SWNs were able to rescue the secondary wall defect in the sndl nstl mutant, indicating that they are functionally interchangeable. Together, our results provide important insight into the complex transcriptional program and the evolutionary mechanism underlying secondary wall biosynthesis, cell wall modification, and programmed cell death in secondary wall-containing cell types.

The miR319-Targeted GhTCP4 Promotes the Transition from Cell Elongation to Wall Thickening in Cotton Fiber

Plant cell growth involves a complex interplay among cell-wall expansion, biosynthesis, and, in specific tissues, secondary cell wall (SCW) deposition, yet the coordination of these processes remains elusive. Cotton fiber cells are developmentally synchronous, highly elongated, and contain nearly pure cellulose when mature. Here, we report that the transcription factor GhTCP4 plays an important role in balancing cotton fiber cell elongation and wall synthesis. During fiber development the expression of miR319 declines while GhTCP4 transcript levels increase, with high levels of the latter promoting SCW deposition. GhTCP4 interacts with a homeobox-containing factor, GhHOX3, and repressing its transcriptional activity. GhTCP4 and GhHOX3 function antagonistically to regulate cell elongation, thereby establishing temporal control of fiber cell transition to the SCW stage. We found that overexpression of GhTCP4A upregulated and accelerated activation of the SCW biosynthetic pathway in fiber cells, as revealed by transcriptome and promoter activity analyses, resulting in shorter fibers with varied lengths and thicker walls. In contrast, GhTCP4 downregulation led to slightly longer fibers and thinner cell walls. The GhHOX3–GhTCP4 complex may represent a general mechanism of cellular development in plants since both are conserved factors in many species, thus providing us a potential molecular tool for the design of fiber traits.

Global identification of genes associated with xylan biosynthesis in cotton fiber

Background:Mature cotton fiber secondary cell wall comprises largely of cellulose(>90%)and small amounts of xylan and lignin.Little is known about the cotton fiber xylan biosynthesis by far.Results:To comprehensively survey xylan biosynthetic genes in cotton fiber,we identified five IRX9,five IRX10,one IRX14,six IRX15,two FRA8,one PARVUS,eight GUX,four GXM,two RWA,two AXY9,13 TBL genes by using phylogenetic analysis coupled with expression profile analysis and co-expression analyses.In addition,we also identified two GT61 members,two GT47 members,and two DUF579 family members whose homologs in Arabidopsis were not functionally characterized.These 55 genes were regarded as the most probable genes to be involved in fiber xylan biosynthesis.Further complementation analysis indicated that one IRX10 like and two FRA8 related genes were able to partially recover the irregular xylem phenotype conferred by the xylan deficiency in their respective Arabidopsis mutant.We conclude that these genes are functional orthologs of respective genes that are implicated in GX biosynthesis.Conclusion:The list of 55 cotton genes presented here provides not only a solid basis to uncover the biosynthesis of xylan in cotton fiber,but also a genetic resource potentially useful for future studies aiming at fiber improvement via biotechnological approaches.

PagERF81 regulates lignin biosynthesis and xylem cell differentiation in poplar

Lignin is a major component of plant cell walls and is essential for plant growth and development. Lignin biosynthesis is controlled by a hierarchical regulatory network involving multiple transcription factors. In this study, we showed that the gene encoding an APETALA 2/ethylene-responsive element binding factor(AP2/ERF) transcription factor, PagERF81,from poplar 84 K(Populus alba × P. glandulosa) is highly expressed in expanding secondary xylem cells. Two independent homozygous Pagerf81 mutant lines created by gene editing, produced significantly more but smaller vessel cells and longer fiber cells with more lignin in cell walls, while PagERF81 overexpression lines had less lignin,compared to non-transgenic controls. Transcriptome and reverse transcription quantitative PCR data revealed that multiple lignin biosynthesis genes including Cinnamoyl CoA reductase 1(PagCCR1),Cinnamyl alcohol dehydrogenase 6(PagCAD6), and 4-Coumarate-CoA ligase-like 9(Pag4CLL9) were upregulated in Pagerf81 mutants, but down-regulated in PagERF81 overexpression lines. In addition, a transient transactivation assay revealed that PagERF81 repressed the transcription of these three genes.Furthermore, yeast one hybrid and electrophoretic mobility shift assays showed that PagERF81 directly bound to a GCC sequence in the PagCCR1 promoter. No known vessel or fiber cell differentiation related genes were differentially expressed, so the smaller vessel cells and longer fiber cells observed in the Pagerf81 lines might be caused by abnormal lignin deposition in the secondary cell walls. This study provides insight into the regulation of lignin biosynthesis, and a molecular tool to engineer wood with high lignin content, which would contribute to the lignin-related chemical industry and carbon sequestration.

Altered Lignin Biosynthesis Improves Cellulosic Bioethanol Production in Transgenic Maize Plants Down-Regulated for Cinnamyl Alcohol Dehydrogenase

Cinnamyl alcohol dehydrogenase （CAD） is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Trans- genic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alter- ations in cell wall composition. Cell walls of CAD-RNAi stems contain a lignin polymer with a slight reduction in the S-to-G ratio without affecting the total lignin content. In addition, these cell walls accumulate higher levels of cellulose and ara- binoxylans. In contrast, cell walls of CAD-RNAi midribs present a reduction in the total lignin content and of cell wall polysaccharides. In vitro degradability assays showed that, although to a different extent, the changes induced by the repression of CAD activity produced midribs and stems more degradable than wild-type plants. CAD-RNAi plants grown in the field presented a wild-type phenotype and produced higher amounts of dry biomass. Cellulosic bioethanol assays revealed that CAD-RNAi biomass produced higher levels of ethanol compared to wild-type, making CAD a good target to improve both the nutritional and energetic values of maize lignocellulosic biomass.

The role of senescence-associated gene101 (PagSAG101a) in the regulation of secondary xylem formation in poplar

Wood is produced by the accumulation of secondary xylem via proliferation and differentiation of the cambium cells in woody plants. Identifying the regulators involved in this process remains a challenging task. In this study, we isolated Pag SAG101 a,the homolog of Arabidopsis thaliana SAG101, from a hybrid poplar(Populus alba × Populus glandulosa)clone 84 K and investigated its role in secondary xylem development. Pag SAG101 a was expressed predominantly in lignified stems and localized in thenucleus. Compared with non-transgenic 84 K plants,transgenic plants overexpressing Pag SAG101 a displayed increased plant height, internode number,stem diameter, xylem width, and secondary cell wall thickness, while opposite phenotypes were observed for Pag SAG101 a knock-out plants.Transcriptome analyses revealed that differentially expressed genes were enriched for those controlling cambium cell division activity and subsequent secondary cell wall deposition during xylem formation.In addition, the tandem CCCH zinc finger protein Pag C3 H17, which positively regulates secondary xylem width and secondary wall thickening in poplar, could bind to the promoter of Pag SAG101 a and mediate the regulation of xylem differentiation.Our results support that Pag SAG101 a, downstream of Pag C3 H17, functions in wood development.

NARROW AND ROLLED LEAF 2 regulates leaf shape,male fertility, and seed size in rice

Grain yield in rice （Oryza sativa L.） is closely related to leaf and flower development. Coordinative regulation of leaf, pollen, and seed development in rice as a critical biological and agricultural question should be addressed. Here we identified two allelic rice mutants with narrow and semi- rolled leaves, named narrow and rolled leaf2-1 （nrl2-1） and nr12- 2. Map-based molecular cloning revealed that NRL2 encodes a novel protein with unknown biochemical function. The mutation of NRL2 caused pleiotropic effects, including a reduction in the number of longitudinal veins, defective abaxial sclerenchymatous cell differentiation, abnormal tape- tum degeneration and microspore development, and the formation of more slender seeds compared with the wild type （WT）. The NRL2 protein interacted with Rolling-leaf （RL14）, causing the leaves of the nrl2 mutants to have a higher cellulose content and lower Iignin content than the WT, which may have been related to sclerenchymatous cell differentia- tion and tapetum degeneration. Thus, this gene is an essential developmental regulator controlling fundamental cellular and developmental processes, serving as a potential breeding target for high-yielding rice cultivars.

Analysis of Gene Expression Patterns during Seed Coat Development in Arabidopsis

The seed coat is important for embryo protection, seed hydration, and dispersal. Seed coat composition is also of interest to the agricultural sector, since it impacts the nutritional value for humans and livestock alike. Although some seed coat genes have been identified, the developmental pathways controlling seed coat development are not completely elucidated, and a global genetic program associated with seed coat development has not been reported. This study uses a combination of genetic and genomic approaches in Arabidopsis thaliana to begin to address these knowledge gaps. Seed coat development is a complex process whereby the integuments of the ovule differentiate into specialized cell types. In Arabidopsis, the outermost layer of cells secretes mucilage into the apoplast and develops a secondary cell wall known as a columella. The layer beneath the epidermis, the palisade, synthesizes a secondary cell wall on its inner tangential side. The innermost layer （the pigmented layer or endothelium） produces proanthocyanidins that condense into tannins and oxidize, giving a brown color to mature seeds. Genetic separation of these cell layers was achieved using the ap2-7 and tt16-1 mutants, where the epidermis/palisade and the endothelium do not develop respectively. This genetic ablation was exploited to examine the developmental programs of these cell types by isolating and collecting seed coats at key tran- sitions during development and performing global gene expression analysis. The data indicate that the developmental programs of the epidermis and the pigmented layer proceed relatively independently. Global expression datasets that can be used for identification of new gene candidates for seed coat development were generated. These dataset provide a comprehensive expression profile for developing seed coats in Arabidopsis, and should provide a useful resource and reference for other seed systems.