Transcription factor PagMYB31 positively regulates cambium activity and negatively regulates xylem development in poplar.

Wood formation involves consecutive developmental steps, including cell division of vascular cambium, xylem cell expansion, secondary cell wall (SCW) deposition, and programmed cell death. In this study, we identified PagMYB31 as a coordinator regulating these processes in Populus alba × Populus glandulosa and built a PagMYB31-mediated transcriptional regulatory network. PagMYB31 mutation caused fewer layers of cambial cells, larger fusiform initials, ray initials, vessels, fiber and ray cells, and enhanced xylem cell SCW thickening, showing that PagMYB31 positively regulates cambial cell proliferation and negatively regulates xylem cell expansion and SCW biosynthesis. PagMYB31 repressed xylem cell expansion and SCW thickening through directly inhibiting wall-modifying enzyme genes and the transcription factor genes that activate the whole SCW biosynthetic program, respectively. In cambium, PagMYB31 could promote cambial activity through TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)/PHLOEM INTERCALATED WITH XYLEM (PXY) signaling by directly regulating CLAVATA3/ESR-RELATED (CLE) genes, and it could also directly activate WUSCHEL HOMEOBOX RELATED4 (PagWOX4), forming a feedforward regulation. We also observed that PagMYB31 could either promote cell proliferation through the MYB31-MYB72-WOX4 module or inhibit cambial activity through the MYB31-MYB72-VASCULAR CAMBIUM-RELATED MADS2 (VCM2)/PIN-FORMED5 (PIN5) modules, suggesting its role in maintaining the homeostasis of vascular cambium. PagMYB31 could be a potential target to manipulate different developmental stages of wood formation.

Auxin signaling in the cambium promotes tissue adhesion and vascular formation during Arabidopsis graft healing.

The strong ability of plants to regenerate wounds is exemplified by grafting when two plants are cut and joined together to grow as one. During graft healing, tissues attach, cells proliferate, and the vasculatures connect to form a graft union. The plant hormone auxin plays a central role, and auxin-related mutants perturb grafting success. Here, we investigated the role of individual cell types and their response to auxin during Arabidopsis (Arabidopsis thaliana) graft formation. By employing a cell-specific inducible misexpression system, we blocked auxin response in individual cell types using the bodenlos mutation. We found that auxin signaling in procambial tissues was critical for successful tissue attachment and vascular differentiation. In addition, we found that auxin signaling was required for cell divisions of the procambial cells during graft formation. Loss of function mutants in cambial pathways also perturbed attachment and phloem reconnection. We propose that cambial and procambial tissues drive tissue attachment and vascular differentiation during successful grafting. Our study thus refines our knowledge of graft development and furthers our understanding of the regenerative role of the cambium.

Mobile PEAR transcription factors integrate positional cues to prime cambial growth.

Apical growth in plants initiates upon seed germination, whereas radial growth is primed only during early ontogenesis in procambium cells and activated later by the vascular cambium1. Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on2,3. Here we show that in Arabidopsis the initiation of radial growth occurs around early protophloem-sieve-element cell files of the root procambial tissue. In this domain, cytokinin signalling promotes the expression of a pair of mobile transcription factors-PHLOEM EARLY DOF 1 (PEAR1) and PHLOEM EARLY DOF 2 (PEAR2)-and their four homologues (DOF6, TMO6, OBP2 and HCA2), which we collectively name PEAR proteins. The PEAR proteins form a short-range concentration gradient that peaks at protophloem sieve elements, and activates gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by the HD-ZIP III proteins, well-known polarity transcription factors4-the expression of which is concentrated in the more-internal domain of radially non-dividing procambial cells by the function of auxin, and mobile miR165 and miR166 microRNAs. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, and thereby establish a negative-feedback loop that forms a robust boundary that demarks the zone of cell division. Taken together, our data establish that during root procambial development there exists a network in which a module that links PEAR and HD-ZIP III transcription factors integrates spatial information of the hormonal domains and miRNA gradients to provide adjacent zones of dividing and more-quiescent cells, which forms a foundation for further radial growth.

PagJAZ5 regulates cambium activity through coordinately modulating cytokinin concentration and signaling in poplar.

Wood is resulted from the radial growth paced by the division and differentiation of vascular cambium cells in woody plants, and phytohormones play important roles in cambium activity. Here, we identified that PagJAZ5, a key negative regulator of jasmonate (JA) signaling, plays important roles in enhancing cambium cell division and differentiation by mediating cytokinin signaling in poplar 84K (Populus alba × Populus glandulosa). PagJAZ5 is preferentially expressed in developing phloem and cambium, weakly in developing xylem cells. Overexpression (OE) of PagJAZ5m (insensitive to JA) increased cambium activity and xylem differentiation, while jaz mutants showed opposite results. Transcriptome analyses revealed that cytokinin oxidase/dehydrogenase (CKXs) and type-A response regulators (RRs) were downregulated in PagJAZ5m OE plants. The bioactive cytokinins were significantly increased in PagJAZ5m overexpressing plants and decreased in jaz5 mutants, compared with that in 84K plants. The PagJAZ5 directly interact with PagMYC2a/b and PagWOX4b. Further, we found that the PagRR5 is regulated by PagMYC2a and PagWOX4b and involved in the regulation of xylem development. Our results showed that PagJAZ5 can increase cambium activity and promote xylem differentiation through modulating cytokinin level and type-A RR during wood formation in poplar.

A transcriptional repressor HVA regulates vascular bundle formation through auxin transport in Arabidopsis stem.

Vascular bundles transport water and photosynthate to all organs, and increased bundle number contributes to crop lodging resistance. However, the regulation of vascular bundle formation is poorly understood in the Arabidopsis stem. We report a novel semi-dominant mutant with high vascular activity, hva-d, showing increased vascular bundle number and enhanced cambium proliferation in the stem. The activation of a C2H2 zinc finger transcription factor, AT5G27880/HVA, is responsible for the hva-d phenotype. Genetic, biochemical, and fluorescent microscopic analyses were used to dissect the functions of HVA. HVA functions as a repressor and interacts with TOPLESS via the conserved Ethylene-responsive element binding factor-associated Amphiphilic Repression motif. In contrast to the HVA activation line, knockout of HVA function with a CRISPR-Cas9 approach or expression of HVA fused with an activation domain VP16 (HVA-VP16) resulted in fewer vascular bundles. Further, HVA directly regulates the expression of the auxin transport efflux facilitator PIN1, as a result affecting auxin accumulation. Genetics analysis demonstrated that PIN1 is epistatic to HVA in controlling bundle number. This research identifies HVA as a positive regulator of vascular initiation through negatively modulating auxin transport and sheds new light on the mechanism of bundle formation in the stem.

A molecular module connects abscisic acid with auxin signals to facilitate seasonal wood formation in Populus.

Perennial trees have a recurring annual cycle of wood formation in response to environmental fluctuations. However, the precise molecular mechanisms that regulate the seasonal formation of wood remain poorly understood. Our prior study indicates that VCM1 and VCM2 play a vital role in regulating the activity of the vascular cambium by controlling the auxin homoeostasis of the cambium zone in Populus. This study indicates that abscisic acid (ABA) affects the expression of VCM1 and VCM2, which display seasonal fluctuations in relation to photoperiod changes. ABA-responsive transcription factors AREB4 and AREB13, which are predominantly expressed in stem secondary vascular tissue, bind to VCM1 and VCM2 promoters to induce their expression. Seasonal changes in the photoperiod affect the ABA amount, which is linked to auxin-regulated cambium activity via the functions of VCM1 and VCM2. Thus, the study reveals that AREB4/AREB13-VCM1/VCM2-PIN5b acts as a molecular module connecting ABA and auxin signals to control vascular cambium activity in seasonal wood formation.

Gene co-expression network analysis identifies BEH3 as a stabilizer of secondary vascular development in Arabidopsis.

In plants, vascular stem cells located in the cambium continuously undergo self-renewal and differentiation during secondary growth. Recent advancements in cell sorting techniques have enabled access to the transcriptional regulatory framework of cambial cells. However, mechanisms underlying the robust control of vascular stem cells remain unclear. Here, we identified a new cambium-related regulatory module through co-expression network analysis using multiple transcriptome datasets obtained from an ectopic vascular cell transdifferentiation system using Arabidopsis cotyledons, Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL). The cambium gene list included a gene encoding the transcription factor BES1/BZR1 Homolog 3 (BEH3), whose homolog BES1 negatively affects vascular stem cell maintenance. Interestingly, null beh3 mutant alleles showed a large variation in their vascular size, indicating that BEH3 functions as a stabilizer of vascular stem cells. Genetic analysis revealed that BEH3 and BES1 perform opposite functions in the regulation of vascular stem cells and the differentiation of vascular cells in the context of the VISUAL system. At the biochemical level, BEH3 showed weak transcriptional repressor activity and functioned antagonistically to other BES/BZR members by competing for binding to the brassinosteroid response element. Furthermore, mathematical modeling suggested that the competitive relationship between BES/BZR homologs leads to the robust regulation of vascular stem cells.

Network Analysis of Metabolome and Transcriptome Revealed Regulation of Different Nitrogen Concentrations on Hybrid Poplar Cambium Development.

Secondary development is a key biological characteristic of woody plants and the basis of wood formation. Exogenous nitrogen can affect the secondary growth of poplar, and some regulatory mechanisms have been found in the secondary xylem. However, the effect of nitrogen on cambium has not been reported. Herein, we investigated the effects of different nitrogen concentrations on cambium development using combined transcriptome and metabolome analysis. The results show that, compared with 1 mM NH4NO3 (M), the layers of hybrid poplar cambium cells decreased under the 0.15 mM NH4NO3 (L) and 0.3 mM NH4NO3 (LM) treatments. However, there was no difference in the layers of hybrid poplar cambium cells under the 3 mM NH4NO3 (HM) and 5 mM NH4NO3 (H) treatments. Totals of 2365, 824, 649 and 398 DEGs were identified in the M versus (vs.) L, M vs. LM, M vs. HM and M vs. H groups, respectively. Expression profile analysis of the DEGs showed that exogenous nitrogen affected the gene expression involved in plant hormone signal transduction, phenylpropanoid biosynthesis, the starch and sucrose metabolism pathway and the ubiquitin-mediated proteolysis pathway. In M vs. L, M vs. LM, M vs. HM and M vs. H, differential metabolites were enriched in flavonoids, lignans, coumarins and saccharides. The combined analysis of the transcriptome and metabolome showed that some genes and metabolites in plant hormone signal transduction, phenylpropanoid biosynthesis and starch and sucrose metabolism pathways may be involved in nitrogen regulation in cambium development, whose functions need to be verified. In this study, from the point of view that nitrogen influences cambium development to regulate wood formation, the network analysis of the transcriptome and metabolomics of cambium under different nitrogen supply levels was studied for the first time, revealing the potential regulatory and metabolic mechanisms involved in this process and providing new insights into the effects of nitrogen on wood development.

Gibberellin promotes cambium reestablishment during secondary vascular tissue regeneration after girdling in an auxin-dependent manner in Populus.

Secondary vascular tissue (SVT) development and regeneration are regulated by phytohormones. In this study, we used an in vitro SVT regeneration system to demonstrate that gibberellin (GA) treatment significantly promotes auxin-induced cambium reestablishment. Altering GA content by overexpressing or knocking down ent-kaurene synthase (KS) affected secondary growth and SVT regeneration in poplar. The poplar DELLA gene GIBBERELLIC ACID INSENSITIVE (PtoGAI) is expressed in a specific pattern during secondary growth and cambium regeneration after girdling. Overexpression of PtoGAI disrupted poplar growth and inhibited cambium regeneration, and the inhibition of cambium regeneration could be partially restored by GA application. Further analysis of the PtaDR5:GUS transgenic plants, the localization of PIN-FORMED 1 (PIN1) and the expression of auxin-related genes found that an additional GA treatment could enhance the auxin response as well as the expression of PIN1, which mediates auxin transport during SVT regeneration. Taken together, these findings suggest that GA promotes cambium regeneration by stimulating auxin signal transduction.

Translational profile of developing phellem cells in Arabidopsis thaliana roots.

The phellem is a specialized boundary tissue providing the first line of defense against abiotic and biotic stresses in organs undergoing secondary growth. Phellem cells undergo several differentiation steps, which include cell wall suberization, cell expansion, and programmed cell death. Yet, the molecular players acting particularly in phellem cell differentiation remain poorly described, particularly in the widely used model plant Arabidopsis thaliana. Using specific marker lines we followed the onset and progression of phellem differentiation in A. thaliana roots and further targeted the translatome of newly developed phellem cells using translating ribosome affinity purification followed by mRNA sequencing (TRAP-SEQ). We showed that phellem suberization is initiated early after phellogen (cork cambium) division. The specific translational landscape was organized in three main domains related to energy production, synthesis and transport of cell wall components, and response to stimulus. Novel players in phellem differentiation related to suberin monomer transport and assembly as well as novel transcription regulators were identified. This strategy provided an unprecedented resolution of the translatome of developing phellem cells, giving a detailed and specific view on the molecular mechanisms acting on cell differentiation in periderm tissues of the model plant Arabidopsis.

Sucrose Signaling Contributes to the Maintenance of Vascular Cambium by Inhibiting Cell Differentiation.

Plants produce sugars by photosynthesis and use them for growth and development. Sugars are transported from source-to-sink organs via the phloem in the vasculature. It is well known that vascular development is precisely controlled by plant hormones and peptide hormones. However, the role of sugars in the regulation of vascular development is poorly understood. In this study, we examined the effects of sugars on vascular cell differentiation using a vascular cell induction system named 'Vascular Cell Induction Culture System Using Arabidopsis Leaves' (VISUAL). We found that sucrose has the strongest inhibitory effect on xylem differentiation, among several types of sugars. Transcriptome analysis revealed that sucrose suppresses xylem and phloem differentiation in cambial cells. Physiological and genetic analyses suggested that sucrose might function through the BRI1-EMS-SUPPRESSOR1 transcription factor, which is the central regulator of vascular cell differentiation. Conditional overexpression of cytosolic invertase led to a decrease in the number of cambium layers due to an imbalance between cell division and differentiation. Taken together, our results suggest that sucrose potentially acts as a signal that integrates environmental conditions with the developmental program.

Genes expression profiles in vascular cambium of Eucalyptus urophylla × Eucalyptus grandis at different ages.

BACKGROUND: Wood is a secondary xylem generated by vascular cambium. Vascular cambium activities mainly include cambium proliferation and vascular tissue formation through secondary growth, thereby producing new secondary phloem inward and secondary xylem outward and leading to continuous tree thickening and wood formation. Wood formation is a complex biological process, which is strictly regulated by multiple genes. Therefore, molecular level research on the vascular cambium of different tree ages can lead to the identification of both key and related genes involved in wood formation and further explain the molecular regulation mechanism of wood formation. RESULTS: In the present study, RNA-Seq and Pac-Bio Iso-Seq were used for profiling gene expression changes in Eucalyptus urophylla × Eucalyptus grandis (E. urograndis) vascular cambium at four different ages. A total of 59,770 non-redundant transcripts and 1892 differentially expressed genes (DEGs) were identified. The expression trends of the DEGs related to cell division and differentiation, cell wall biosynthesis, phytohormone, and transcription factors were analyzed. The DEGs encoding expansin, kinesin, cycline, PAL, GRP9, KNOX, C2C2-dof, REV, etc., were highly expressed in E. urograndis at three years old, leading to positive effects on growth and development. Moreover, some gene family members, such as NAC, MYB, HD-ZIP III, RPK, and RAP, play different regulatory roles in wood formation because of their sophisticated transcriptional network and function redundantly. CONCLUSIONS: These candidate genes are a potential resource to further study wood formation, especially in fast-growing and adaptable eucalyptus. The results may also serve as a basis for further research to unravel the molecular mechanism underlying wood formation.

Strigolactone signaling regulates cambial activity through repression of WOX4 by transcription factor BES1.

During secondary growth, meristematic cells in the cambium can either proliferate to maintain the stem cell population or differentiate into xylem or phloem. The balance between these two developmental trajectories is tightly regulated by many environmental and endogenous cues. Strigolactones (SLs), a class of plant hormones, were previously reported to regulate secondary growth by promoting cambium activity. However, the underlying molecular mechanisms of SL action in plant secondary growth are not well understood. We performed histological, genetic, and biochemical analyses using genetic materials in Arabidopsis (Arabidopsis thaliana) with altered activity of the transcription factors BRI1-EMS-SUPPRESSOR1 (BES1) or WUSCHEL-related HOMEOBOX4 (WOX4) or lacking MORE AXILLARY SHOOT2 (MAX2), a key positive component in the SL signaling pathway. We found that BES1, a downstream regulator in the SL signaling pathway that promotes shoot branching and xylem differentiation, also inhibits WOX4 expression, a key regulator of cambium cell division in the intercellular TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)-TDIF RECEPTOR (TDR) signaling pathway. The antagonistic roles of BES1 and WOX4 in the regulation of cambium activity may integrate intercellular TDIF signals to efficiently and bidirectionally modulate cambium cell proliferation and differentiation. As both BES1 and WOX4 are widely involved in various endogenous signals and responses to environmental stimuli, these findings may provide insight into the dynamic regulation of cambium development.

Water lily (Nymphaea thermarum) genome reveals variable genomic signatures of ancient vascular cambium losses.

For more than 225 million y, all seed plants were woody trees, shrubs, or vines. Shortly after the origin of angiosperms ∼140 million y ago (MYA), the Nymphaeales (water lilies) became one of the first lineages to deviate from their ancestral, woody habit by losing the vascular cambium, the meristematic population of cells that produces secondary xylem (wood) and phloem. Many of the genes and gene families that regulate differentiation of secondary tissues also regulate the differentiation of primary xylem and phloem, which are produced by apical meristems and retained in nearly all seed plants. Here, we sequenced and assembled a draft genome of the water lily Nymphaea thermarum, an emerging system for the study of early flowering plant evolution, and compared it to genomes from other cambium-bearing and cambium-less lineages (e.g., monocots and Nelumbo). This revealed lineage-specific patterns of gene loss and divergence. Nymphaea is characterized by a significant contraction of the HD-ZIP III transcription factors, specifically loss of REVOLUTA, which influences cambial activity in other angiosperms. We also found the Nymphaea and monocot copies of cambium-associated CLE signaling peptides display unique substitutions at otherwise highly conserved amino acids. Nelumbo displays no obvious divergence in cambium-associated genes. The divergent genomic signatures of convergent loss of vascular cambium reveals that even pleiotropic genes can exhibit unique divergence patterns in association with independent events of trait loss. Our results shed light on the evolution of herbaceousness-one of the key biological innovations associated with the earliest phases of angiosperm evolution.

The BOP-type co-transcriptional regulator NODULE ROOT1 promotes stem secondary growth of the tropical Cannabaceae tree Parasponia andersonii.

Tree stems undergo a massive secondary growth in which secondary xylem and phloem tissues arise from the vascular cambium. Vascular cambium activity is driven by endogenous developmental signalling cues and environmental stimuli. Current knowledge regarding the genetic regulation of cambium activity and secondary growth is still far from complete. The tropical Cannabaceae tree Parasponia andersonii is a non-legume research model of nitrogen-fixing root nodulation. Parasponia andersonii can be transformed efficiently, making it amenable for CRISPR-Cas9-mediated reverse genetics. We considered whether P. andersonii also could be used as a complementary research system to investigate tree-related traits, including secondary growth. We established a developmental map of stem secondary growth in P. andersonii plantlets. Subsequently, we showed that the expression of the co-transcriptional regulator PanNODULE ROOT1 (PanNOOT1) is essential for controlling this process. PanNOOT1 is orthologous to Arabidopsis thaliana BLADE-ON-PETIOLE1 (AtBOP1) and AtBOP2, which are involved in the meristem-to-organ-boundary maintenance. Moreover, in species forming nitrogen-fixing root nodules, NOOT1 is known to function as a key nodule identity gene. Parasponia andersonii CRISPR-Cas9 loss-of-function Pannoot1 mutants are altered in the development of the xylem and phloem tissues without apparent disturbance of the cambium organization and size. Transcriptomic analysis showed that the expression of key secondary growth-related genes is significantly down-regulated in Pannoot1 mutants. This allows us to conclude that PanNOOT1 positively contributes to the regulation of stem secondary growth. Our work also demonstrates that P. andersonii can serve as a tree research system.

A Constitutively Active Cytokinin Receptor Variant Increases Cambial Activity and Stem Growth in Poplar.

The cambial meristem is responsible for bark and wood formation in woody plants. The activity of the cambial meristem is controlled by various factors; one of them is the plant hormone cytokinin. Here, we have explored different approaches to genetically engineering cambial activity in poplar plants by the ectopic expression of a cytokinin biosynthesis gene with enhanced activity (named ROCK4) or of a gene encoding a constitutively active cytokinin receptor variant (ROCK3). Both genes are derived from Arabidopsis thaliana and were expressed in poplar trees under the control of their own promoter or the cambium-specific pHB8 promoter. pIPT3:ROCK4- and pHB8:ROCK4-expressing plants were smaller than wild-type plants and formed more lateral branches; pHB8:ROCK4 transgenic plants additionally showed an increased stem diameter. In contrast, pAHK3:ROCK3- and pHB8:ROCK3-expressing plants grew taller than wild type without an altered branching pattern and formed more cambial cells, leading to increased radial stem growth. The effectivity of ROCK3 when expressed in either secondary phloem cells or in cambial cells is consistent with a dual, tissue-autonomous and non-autonomous activity of cytokinin in regulating cambial activity. We propose ROCK3 as a novel gene to enhance biomass formation in woody plants.

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 PagSAG101a, the homolog of Arabidopsis thaliana SAG101, from a hybrid poplar (Populus alba × Populus glandulosa) clone 84K and investigated its role in secondary xylem development. PagSAG101a was expressed predominantly in lignified stems and localized in the nucleus. Compared with non-transgenic 84K plants, transgenic plants overexpressing PagSAG101a displayed increased plant height, internode number, stem diameter, xylem width, and secondary cell wall thickness, while opposite phenotypes were observed for PagSAG101a 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 PagC3H17, which positively regulates secondary xylem width and secondary wall thickening in poplar, could bind to the promoter of PagSAG101a and mediate the regulation of xylem differentiation. Our results support that PagSAG101a, downstream of PagC3H17, functions in wood development.

Identification of regulatory factors promoting embryogenic callus formation in barley through transcriptome analysis.

BACKGROUND: Barley is known to be recalcitrant to tissue culture, which hinders genetic transformation and its biotechnological application. To date, the ideal explant for transformation remains limited to immature embryos; the mechanism underlying embryonic callus formation is elusive. RESULTS: This study aimed to uncover the different transcription regulation pathways between calli formed from immature (IME) and mature (ME) embryos through transcriptome sequencing. We showed that incubation of embryos in an auxin-rich medium caused dramatic changes in gene expression profiles within 48 h. Overall, 9330 and 11,318 differentially expressed genes (DEGs) were found in the IME and ME systems, respectively. 3880 DEGs were found to be specific to IME_0h/IME_48h, and protein phosphorylation, regulation of transcription, and oxidative-reduction processes were the most common gene ontology categories of this group. Twenty-three IAA, fourteen ARF, eight SAUR, three YUC, and four PIN genes were found to be differentially expressed during callus formation. The effect of callus-inducing medium (CIM) on IAA genes was broader in the IME system than in the ME system, indicating that auxin response participates in regulating cell reprogramming during callus formation. BBM, LEC1, and PLT2 exhibited a significant increase in expression levels in the IME system but were not activated in the ME system. WUS showed a more substantial growth trend in the IME system than in the ME system, suggesting that these embryonic, shoot, and root meristem genes play crucial roles in determining the acquisition of competency. Moreover, epigenetic regulators, including SUVH3A, SUVH2A, and HDA19B/703, exhibited differential expression patterns between the two induction systems, indicating that epigenetic reprogramming might contribute to gene expression activation/suppression in this process. Furthermore, we examined the effect of ectopic expression of HvBBM and HvWUS on Agrobacterium-mediated barley transformation. The transformation efficiency in the group expressing the PLTPpro:HvBBM + Axig1pro:HvWUS construct was increased by three times that in the control (empty vector) because of enhanced plant regeneration capacity. CONCLUSIONS: We identified some regulatory factors that might contribute to the differential responses of the two explants to callus induction and provide a promising strategy to improve transformation efficiency in barley.

Cytokinin signaling localized in phloem noncell-autonomously regulates cambial activity during secondary growth of Populus stems.

The regulation of cytokinin on secondary vascular development has been uncovered by modulating cytokinin content. However, it remains unclear how cytokinin enriched in developing secondary phloem regulates cambium activity in poplar. Here, we visualized the gradient distribution of cytokinin with a peak in the secondary phloem of poplar stem via immunohistochemical imaging, and determined the role of phloem-located cytokinin signaling during wood formation. We generated transgenic poplar harboring cytokinin oxidase/dehydrogenase (CKX)2, a gene encoding a cytokinin degrading enzyme, driven by the phloem-specific CLE41b promoter, indicating that the disruption of the cytokinin gradient pattern restricts the cambial activity. The RNA interference-based knockdown of the histidine kinase (HK) genes encoding cytokinin receptors specifically in secondary phloem significantly compromised the division activity of cambial cells, whereas the phloem-specific expression of a type-B response regulator (RR) transcription factor stimulated cambial proliferation, providing evidence for the noncell-autonomous regulation of local cytokinin signaling on the cambial activity. Moreover, the cambium-specific knockdown of HKs also led to restricted cambial activity, and the defects were aggravated by the reduced cytokinin accumulation. Our results showed that local cytokinin signaling in secondary phloem regulates cambial activity noncell-autonomously, and coordinately with its local signaling in cambium.

Auxin signaling and vascular cambium formation enable storage metabolism in cassava tuberous roots.

Cassava storage roots are among the most important root crops worldwide, and represent one of the most consumed staple foods in sub-Saharan Africa. The vegetatively propagated tropical shrub can form many starchy tuberous roots from its stem. These storage roots are formed through the activation of secondary root growth processes. However, the underlying genetic regulation of storage root development is largely unknown. Here we report distinct structural and transcriptional changes occurring during the early phases of storage root development. A pronounced increase in auxin-related transcripts and the transcriptional activation of secondary growth factors, as well as a decrease in gibberellin-related transcripts were observed during the early stages of secondary root growth. This was accompanied by increased cell wall biosynthesis, most notably increased during the initial xylem expansion within the root vasculature. Starch storage metabolism was activated only after the formation of the vascular cambium. The formation of non-lignified xylem parenchyma cells and the activation of starch storage metabolism coincided with increased expression of the KNOX/BEL genes KNAT1, PENNYWISE, and POUND-FOOLISH, indicating their importance for proper xylem parenchyma function.