Ultrastructural Study of Secondary Wall Formation in the Stem Fiber of Phyllostachys pubescens

Ultrastructural changes in secondary wall formation of Phyllostachys pubescens Mazel fiber were investigated with transmission electron microscopy. Fiber developed initially with the elongation of cells containing ribosomes, mitochondria and Golgi bodies in the dense cytoplasm. During the wall thickening, the number of rough endoplasmic reticulum and Golgi bodies increased apparently. There were two kinds of Golgi vesicles, together with the ones from endoplasmic reticulum formed transport vesicles. Many microtubules were arranged parallel to the long axis of the cell adjacent to the plasmalemma. Along with the further development of fiber, polylamellate structure of the secondary wall appeared, with concurrent agglutination of chromatin in the nucleus, swelling and disintegration of organelles, while cortical microtubules were still arranged neatly against the inner side of plasmalemma. Lomasomes could be observed between the wall and plasmalemma. The results indicated that the organelles, such as Golgi bodies together with small vesicles, rough endoplasmic reticulum and lomasomes, played the key role in the thickening and lignification of the secondary wall of bamboo fiber, though cortical microtubules were correlative with the process as well.

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.

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.

Upregulation of the glycine-rich protein-encoding gene GhGRPL enhances plant tolerance to abiotic and biotic stressors by promoting secondary cell wall development

Abiotic and biotic stressors adversely affect plant survival,biomass generation,and crop yields.As the global availability of arable land declines and the impacts of global warming intensify,such stressors may have increasingly pronounced effects on agricultural productivity.Currently,researchers face the overarching challenge of comprehensively enhancing plant resilience to abiotic and biotic stressors.The secondary cell wall plays a crucial role in bolstering the stress resistance of plants.To increase plant resistance to stress through genetic manipulation of the secondary cell wall,we cloned a cell wall protein designated glycine-rich protein-like(GhGRPL)from cotton fibers,and found that it is specifically expressed during the period of secondary cell wall biosynthesis.Notably,this protein differs from its Arabidopsis homolog,AtGRP,since its glycine-rich domain is deficient in glycine residues.GhGRPL is involved in secondary cell wall deposition.Upregulation of GhGRPL enhances lignin accumulation and,consequently,the thickness of the secondary cell walls,thereby increasing the plant’s resistance to abiotic stressors,such as drought and salinity,and biotic threats,including Verticillium dahliae infection.Conversely,interference with GhGRPL expression in cotton reduces lignin accumulation and compromises that resistance.Taken together,our findings elucidate the role of GhGRPL in regulating secondary cell wall development through its influence on lignin deposition,which,in turn,reinforces cell wall robustness and impermeability.These findings highlight the promising near-future prospect of adopting GhGRPL as a viable,effective approach for enhancing plant resilience to abiotic and biotic stress factors.

An Uncanonical CCCH-Tandem Zinc-Finger Protein Represses Secondary Wall Synthesis and Controls Mechanical Strength in Rice

Secondary walls, which represent the bulk of biomass, have a large impact on plant growth and adaptation to environments. Secondary wall synthesis is switched and regulated by a sophisticated signaling transduction network. However, there is limited understanding of these regulatory pathways. Here, we report that ILAl-interacting protein 4 （lIP4） can repress secondary wall synthesis, lIP4 is a phosphorylation sub- strate of an Raf-like MAPKKK, but its function is unknown. By generating lip4 mutants and relevant transgenic plants, we found that lesions in lIP4 enhance secondary wall formation. Gene expression and transactivation activity assays revealed that lIP4 negatively regulates the expression of MYB61 and CESAs but does not bind their promoters, lIP4 interacts with NAC29/NAC31, the upstream regulators of secondary wall synthesis, and suppresses the downstream regulatory pathways in plants. Mutagenesis analyses showed that phosphomimic UP4 proteins translocate from the nucleus to the cytoplasm, which releases interacting NACs and attenuates its repression function. Moreover, we revealed that liPs are evolutionarily conserved and share unreported CCCH motifs, referred to as uncanonical CCCH-tandem zinc-finger proteins. Collectively, our study provides mechanistic insights into the control of secondary wall synthesis and presents an opportunity for improving relevant agronomic traits in crops.

Identification of novel cis-elements bound by BplMYB46 involved in abiotic stress responses and secondary wall deposition

Transcription factors （TFs） play vital roles in various biological processes by binding to cis-acting elements to control expressions of their target genes. The MYB TF BplMYB46, from Betula platyphylla, is involved in abiotic stress responses and secondary wall deposition. In the present study, we used a TF-centered yeast onehybrid technology （TF-centered YIH） to identify the cis- acting elements bound by BplMYB46. We screened a shortinsert random library and identified three cis-elements bound by BplMYB46： an E-box （CA（A/T/C）（A/G/C）TG） and two novel motifs, a TO-box （T（GIA）TCG（C/G）） and a GT-box （A（G/T）T（AIC）GT（T/G）C）. Chromatin immunoprecipitation （CHIP） and effector-reporter coexpression assays inNicotiana tabacum confirmed binding of BplMYB46 to the TC-box, GT-box, and E-box motifs in the promoters of the phenylalanine ammonia lyase （PAL）, peroxidase （POD）, and superoxide dismutase （SOD） genes, which function in abiotic stress tolerance and secondary wall biosynthesis. This finding improves our understanding of potential regulatory mechanisms in the response to abiotic stress and secondary wall deposition of BplMYB46 in B. platyphylla.

Alteration in Secondary Wall Deposition by Overexpression of the Fragile Fiber1 Kinesin-Like Protein in Arabidopsis

Secondary walls in fibers and vessels are typically deposited in three distinct layers, which are formed by the successive re-orientation of cellulose microfibrils. Although cortical microtubules have been implicated in this process, the underlying mechanisms for the formation of three distinct wall layers are not known. The Fragile Fiber1 （FRA1） kinesin-like protein has been previously shown to be involved in the oriented deposition of cellulose microfibrils and important for cell wall strength in Arabidopsis thaliana. In the present report, we investigated the expression pattern of the FRA 1 gene and studied the effects of FRA1 overexpression on secondary wall deposition. The FRAI gene was found to be expressed not only in cells undergoing secondary wall deposition including developing interfascicular fibers and xylem cells, but also in dividing cells and expanding/elongating parenchyma cells. Overexpression of FRA1 caused a severe reduction in the thickness of secondary walls in interfascicular fibers and deformation of vessels, which are accompanied with a marked decrease in stem strength. Close examination of secondary walls revealed that unlike the wild-type walls having three typical layers with the middle layer being the thickest, the secondary walls in FRA1 overexpressors exhibited an increased number of layers, all of which had a similar width. Together, these results provide further evidence implicating an important role of the FRA1 kinesin-like protein in the ordered deposition of secondary walls, which determines the strength of fibers and vessels.

Phylogenetically Distinct Cellulose Synthase Genes Support Secondary Wall Thickening in Arabidopsis Shoot Trichomes and Cotton Fiber

Through exploring potential analogies between cotton seed trichomes （or cotton fiber） and arabidopsis shoot trichomes we discovered that CesAs from either the primary or secondary wall phylogenetic clades can support secondary wall thickening. CesA genes that typically support primary wall synthesis, AtCesA 1,2,3,5, and 6, underpin expansion and secondary wall thickening of arabidopsis shoot trichomes. In contrast, apparent orthologs of CesA genes that support secondary wall synthesis in arabidopsis xylem, AtCesA4,7, and 8, are up-regulated for cotton fiber secondary wall deposition. These conclusions arose from： （a） analyzing the expression of CesA genes in arabidopsis shoot trichomes; （b） observing birefringent secondary walls in arabidopsis shoot trichomes with mutations in AtCesA4, 7, or 8; （c） assaying up-regulated genes during different stages of cotton fiber development; and （d） comparing genes that were co.expressed with primary or secondary wall CesAs in arabidopsis with genes up- regulated in arabidopsis trichomes, arabidopsis secondary xylem, or cotton fiber during primary or secondary wall deposition. Cumulatively, the data show that： （a） the xylem of arabidopsis provides the best model for secondary wall cellulose synthesis in cotton fiber; and （b） CesA genes within a ＂cell wall toolbox＂ are used in diverse ways for the construction of particular specialized cell walls.

Identification and Profiling of Known and Novel Fiber MicroRNAs during the Secondary Wall Thickening Stage in Cotton(Gossypium hirsutum) via High-Throughput Sequencing

Upland cotton （Gossypium hirsutum L.） is an allotetraploid species originated from interspecific hybridization between AA-genome diploid （G. arboretum） and DD-genome diploid （G. raimondii） （Wendel et al., 1992）. Cotton fibers are single-celled trichomes that emerge from the ovule epidermal cells. Indexed by the number of days post-anthesis （dpa）, fiber morphogenesis includes four distinct but overlapping steps： initiation （0-3 dpa）, elongation （3-20 dpa）, secondary cell wall thickening （15-45 dpa） and maturation （40-60 dpa） （Yang et al., 2008, Du et al., 2013）. The efficiency and duration of each morphogenesis stage is important to the quality attributes of the mature fiber. Cell elongation is critical for fiber length, whereas secondary cell wall thickening is important for fiber fineness and strength （Meinert and Delmer, 1977）.

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.

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.

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-

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.

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.

Diurnal Periodicity of the Expression of Genes Involved in Monolignol Biosynthesis in Differentiating Xylem of Cryptomeria japonica

The cell wall in wood is mainly composed of three components: cellulose, hemicellulose and lignin. According to electron microscopy observations of the innermost surface of cell walls in the tracheids of Cryptomeria japonica , cellulose microfibrils are deposited during the day and a matrix containing hemicellulose is deposited at night. This indicates that the deposition of cell wall components exhibits diurnal periodicity. To gain new insights into the diurnal periodicity of lignin deposition not revealed by microscopic observations, we examined diurnal fluctuations in the expression of genes involved in monolignol biosynthesis in C. japonica saplings grown in the field and in growth chambers under a 12 h light/dark cycle. In the field experiment, two gene expression peaks were observed daily, at dusk and dawn. In the growth chamber experiment, two daily peaks were observed 0 h and 6 - 9 h after the light switched on.

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.

Cotton GhMYB7 is predominantly expressed in developing fibers and regulates secondary cell wall biosynthesis in transgenic Arabidopsis

The secondary cell wall in mature cotton fibers contains over 90%cellulose with low quantities of xylan and lignin.However,little is known regarding the regulation of secondary cell wall biosynthesis in cotton fibers.In this study,we characterized an R2R3-MYB transcription factor,Gh MYB7,in cotton.Gh MYB7 is expressed at a high level in developing fibers and encodes a MYB protein that is targeted to the cell nucleus and has transcriptional activation activity.Ectopic expression of Gh MYB7 in Arabidopsis resulted in small,curled,dark green leaves and also led to shorter inflorescence stems.A cross-sectional assay of basal stems revealed that cell wall thickness of vessels and interfascicular fibers was higher in transgenic lines overexpressing Gh MYB7 than in the wild type.Constitutive expression of Gh MYB7 in Arabidopsis activated the expression of a suite of secondary cell wall biosynthesis-related genes(including some secondary cell wall-associated transcription factors),leading to the ectopic deposition of cellulose and lignin.The ectopic deposition of secondary cell walls may have been initiated before the cessation of cell expansion.Moreover,Gh MYB7 was capable of binding to the promoter regions of At SND1 and At Ces A4,suggesting that Gh MYB7 may function upstream of NAC transcription factors.Collectively,these findings suggest that Gh MYB7 is a potential transcriptional activator,which may participate in regulating secondary cell wall biosynthesis of cotton fibers.

Transition of primary to secondary cell wall synthesis

The construction of a secondary cell wall is an important and necessary developmental decision that sup- ports cell function and plant stature. Unlike the primary cell walls, which are initiated during cell division and develop along with the expansion of the cells, secondary cell walls are constructed after the cells have stopped growing. Hence, the transition from primary to secondary wall synthesis marks an important and distinct metabolic investment by the plant. This transition requires a coordi- nated change of a plethora of cellular processes, including hormonal, transcriptional and post-transcriptional activi- ties, metabolic flux re-distributions and enzymatic activities. In this review, we briefly summarize the hormonal and transcriptional control of the primary to secondary wall transition, and highlight important gaps in our under- standing of the metabolic framework that support the transition. Several tools that may aid in future research efforts to better understand the changes in cell wall synthesis during the trans-differentiation are also discussed.

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.