Cytokinin signaling regulates cambial development in poplar.

Although a substantial proportion of plant biomass originates from the activity of vascular cambium, the molecular basis of radial plant growth is still largely unknown. To address whether cytokinins are required for cambial activity, we studied cytokinin signaling across the cambial zones of 2 tree species, poplar (Populus trichocarpa) and birch (Betula pendula). We observed an expression peak for genes encoding cytokinin receptors in the dividing cambial cells. We reduced cytokinin levels endogenously by engineering transgenic poplar trees (P. tremula x tremuloides) to express a cytokinin catabolic gene, Arabidopsis CYTOKININ OXIDASE 2, under the promoter of a birch CYTOKININ RECEPTOR 1 gene. Transgenic trees showed reduced concentration of a biologically active cytokinin, correlating with impaired cytokinin responsiveness. In these trees, both apical and radial growth was compromised. However, radial growth was more affected, as illustrated by a thinner stem diameter than in WT at same height. To dissect radial from apical growth inhibition, we performed a reciprocal grafting experiment. WT scion outgrew the diameter of transgenic stock, implicating cytokinin activity as a direct determinant of radial growth. The reduced radial growth correlated with a reduced number of cambial cell layers. Moreover, expression of a cytokinin primary response gene was dramatically reduced in the thin-stemmed transgenic trees. Thus, a reduced level of cytokinin signaling is the primary basis for the impaired cambial growth observed. Together, our results show that cytokinins are major hormonal regulators required for cambial development.

Transcriptional regulatory framework for vascular cambium development in Arabidopsis roots.

Vascular cambium, a lateral plant meristem, is a central producer of woody biomass. Although a few transcription factors have been shown to regulate cambial activity1, the phenotypes of the corresponding loss-of-function mutants are relatively modest, highlighting our limited understanding of the underlying transcriptional regulation. Here, we use cambium cell-specific transcript profiling followed by a combination of transcription factor network and genetic analyses to identify 62 new transcription factor genotypes displaying an array of cambial phenotypes. This approach culminated in virtual loss of cambial activity when both WUSCHEL-RELATED HOMEOBOX 4 (WOX4) and KNOTTED-like from Arabidopsis thaliana 1 (KNAT1; also known as BREVIPEDICELLUS) were mutated, thereby unlocking the genetic redundancy in the regulation of cambium development. We also identified transcription factors with dual functions in cambial cell proliferation and xylem differentiation, including WOX4, SHORT VEGETATIVE PHASE (SVP) and PETAL LOSS (PTL). Using the transcription factor network information, we combined overexpression of the cambial activator WOX4 and removal of the putative inhibitor PTL to engineer Arabidopsis for enhanced radial growth. This line also showed ectopic cambial activity, thus further highlighting the central roles of WOX4 and PTL in cambium development.

BpMADS4 has a central role in inflorescence initiation in silver birch (Betula pendula).

Acceleration of flowering would be beneficial for breeding trees with a long juvenile phase; conversely, inhibition of flowering would prevent the spread of transgenes from the genetically modified trees. We have previously isolated and characterized several MADS genes from silver birch (Betula pendula Roth). In this study, we investigated the more detailed function of one of them, BpMADS4, a member of the APETALA1/FRUITFULL group of MADS genes. The expression of BpMADS4 starts at very early stage of the male and female inflorescence development and the activity is high in the apex of the developing inflorescence. Later, some expression is detected in the bracts and in the flower initials. Ectopic expression of BpMADS4 accelerates flowering dramatically in normally flowering clones and also in the early-flowering birch clone, in which the earliest line flowered about 11 days after rooting, when the saplings were only 3 cm high. The birches transformed with the BpMADS4 antisense construct showed remarkable delay in flowering and the number of flowering individuals was reduced. Two of the transformed lines did not show any signs of flower development during our 2-year study, whereas all the control plants formed inflorescences within 107 days. Our results show that BpMADS4 has a critical role in the initiation of birch inflorescence development and that BpMADS4 seems to be involved in the transition from vegetative to reproductive development. Therefore, BpMADS4 provides a promising tool for the genetic enhancement of forest trees.

Cytokinin and Auxin Display Distinct but Interconnected Distribution and Signaling Profiles to Stimulate Cambial Activity.

Despite the crucial roles of phytohormones in plant development, comparison of the exact distribution profiles of different hormones within plant meristems has thus far remained scarce. Vascular cambium, a wide lateral meristem with an extensive developmental zonation, provides an optimal system for hormonal and genetic profiling. By taking advantage of this spatial resolution, we show here that two major phytohormones, cytokinin and auxin, display different yet partially overlapping distribution profiles across the cambium. In contrast to auxin, which has its highest concentration in the actively dividing cambial cells, cytokinins peak in the developing phloem tissue of a Populus trichocarpa stem. Gene expression patterns of cytokinin biosynthetic and signaling genes coincided with this hormonal gradient. To explore the functional significance of cytokinin signaling for cambial development, we engineered transgenic Populus tremula × tremuloides trees with an elevated cytokinin biosynthesis level. Confirming that cytokinins function as major regulators of cambial activity, these trees displayed stimulated cambial cell division activity resulting in dramatically increased (up to 80% in dry weight) production of the lignocellulosic trunk biomass. To connect the increased growth to hormonal status, we analyzed the hormone distribution and genome-wide gene expression profiles in unprecedentedly high resolution across the cambial zone. Interestingly, in addition to showing an elevated cambial cytokinin content and signaling level, the cambial auxin concentration and auxin-responsive gene expression were also increased in the transgenic trees. Our results indicate that cytokinin signaling specifies meristematic activity through a graded distribution that influences the amplitude of the cambial auxin gradient.

AINTEGUMENTA and the D-type cyclin CYCD3;1 regulate root secondary growth and respond to cytokinins.

Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT) and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism.

Three MADS-box genes similar to APETALA1 and FRUITFULL from silver birch (Betula pendula).

Despite intensive research on genetic regulation of flower development there are still only a few studies on the early phases of this process in perennial plants like trees. The aim of this study has been to identify genes that regulate early stages of inflorescence development in silver birch (Betula pendula Roth) and to follow the expression of these genes during development of the unisexual birch inflorescences. Here we describe the cloning and characterization of 3 cDNAs representing MADS-box genes designated BpMADS3, BpMADS4 and BpMADS5, all belonging to the AP1/SQUA group of plant MADS-box genes. According to RNA blot analysis, all 3 genes are active during the development of both male and female inflorescences. However, differences in patterns of expression suggest that they play different roles. BpMADS3 is most similar in sequence to AP1 and SQUA, but it seems to have the highest expression at late developmental stages. BpMADS4 is most similar in sequence to the Arabidopsis gene FRUITFULL, but is expressed, in addition to developing inflorescences, in shoots and roots. BpMADS5 is also similar to FRUITFULL; its expression seems to be inflorescence-specific and continues during fruit development. Ectopic expression of either BpMADS3, BpMADS4 or BpMADS5 with the CaMV 35S promoter in tobacco results in extremely early flowering. All of these birch genes seem to act early during the transition to reproductive phase and might be involved in the determination of the identity of the inflorescence or flower meristem. They could apparently be used to accelerate flowering in various plant species.

Functional characterization of SEPALLATA3 and AGAMOUS orthologues in silver birch.

The development of flowers is regulated by a complex network of transcriptional activators and repressors, many of which belong to the MADS box gene family. In this study, we describe two MADS box genes of silver birch (Betula pendula Roth), BpMADS1 and BpMADS6, which are similar to SEPALLATA3 and AGAMOUS in Arabidopsis thaliana, respectively. In situ hybridization showed that BpMADS1 was expressed in the inflorescence meristem at a very early stage, but not later. Both genes were expressed in developing carpels, ovules and stamens but not in tepals or scales. Ectopic expression of BpMADS1 in Arabidopsis resulted in a reduced number of floral organs or whole whorls and in petaloid or carpelloid sepals, a phenotype reminiscent of that of fil mutants. 35S::BpMADS6 caused very early flowering in Arabidopsis. In tobacco, both 35S::BpMADS1 and 35S::BpMADS6 accelerated flowering and, in addition, 35S::BpMADS6 caused changes in sepals and petals. In some transgenic birch plants, 35S::BpMADS1 antisense resulted in the development of both male and female organs in the axil of a single bract and in a change of some inflorescences into vegetative shoots. In two plants, either 35S::BpMADS6 sense or antisense constructs resulted in an increase in the number of tepals and in complete lack of stamens in some male inflorescences. These results suggest that BpMADS1 participates both in inflorescence and in flower formation and BpMADS6 participates in flower formation and that they are functional homologues to SEPALLATA3 and AGAMOUS, respectively.

Arabidopsis as a model for wood formation.

Wood (secondary xylem) is one of the most important sustainable energy sources for humans. Arabidopsis, despite its herbaceous nature, has become an excellent model to study wood formation. Recent progress has shown that conserved molecular mechanisms may exist in herbaceous plants and trees during vascular development and wood formation. Several transcription factor families and plant hormone species as well as other factors contribute to the regulation of xylem development in both Arabidopsis and woody plants. In this review, we highlight how information gained from the analysis of vascular development in Arabidopsis has improved our understanding of wood formation in trees.

Hormone interactions during vascular development.

Vascular tissue in plants is unique due to its diverse and dynamic cellular patterns. Signals controlling vascular development have only recently started to emerge through biochemical, genetic, and genomic approaches in several organisms, such as Arabidopsis, Populus, and Zinnia. These signals include hormones (auxin, brassinosteroids, and cytokinins, in particular), other small regulatory molecules, their transporters, receptors, and various transcriptional regulators. In recent years it has become apparent that plant growth regulators rarely act alone, but rather their signaling pathways are interlocked in complex networks; for example, polar auxin transport (PAT) regulates vascular development during various stages and an emerging theme is its modulation by other growth regulators, depending on the developmental context. Also, several synergistic or antagonistic interactions between various growth regulators have been described. Furthermore, shoot-root interactions appear to be important for this signal integration.

Dual-domain, dual-targeting organellar protein presequences in Arabidopsis can use non-AUG start codons.

The processes accompanying endosymbiosis have led to a complex network of interorganellar protein traffic that originates from nuclear genes encoding mitochondrial and plastid proteins. A significant proportion of nucleus-encoded organellar proteins are dual targeted, and the process by which a protein acquires the capacity for both mitochondrial and plastid targeting may involve intergenic DNA exchange coupled with the incorporation of sequences residing upstream of the gene. We evaluated targeting and sequence alignment features of two organellar DNA polymerase genes from Arabidopsis thaliana. Within one of these two loci, protein targeting appeared to be plastidic when the 5' untranslated leader region (UTR) was deleted and translation could only initiate at the annotated ATG start codon but dual targeted when the 5' UTR was included. Introduction of stop codons at various sites within the putative UTR demonstrated that this region is translated and influences protein targeting capacity. However, no ATG start codon was found within this upstream, translated region, suggesting that translation initiates at a non-ATG start. We identified a CTG codon that likely accounts for much of this initiation. Investigation of the 5' region of other nucleus-encoded organellar genes suggests that several genes may incorporate upstream sequences to influence targeting capacity. We postulate that a combination of intergenic recombination and some relaxation of constraints on translation initiation has acted in the evolution of protein targeting specificity for those proteins capable of functioning in both plastids and mitochondria.

Nuclear genes that encode mitochondrial proteins for DNA and RNA metabolism are clustered in the Arabidopsis genome.

The plant mitochondrial genome is complex in structure, owing to a high degree of recombination activity that subdivides the genome and increases genetic variation. The replication activity of various portions of the mitochondrial genome appears to be nonuniform, providing the plant with an ability to modulate its mitochondrial genotype during development. These and other interesting features of the plant mitochondrial genome suggest that adaptive changes have occurred in DNA maintenance and transmission that will provide insight into unique aspects of plant mitochondrial biology and mitochondrial-chloroplast coevolution. A search in the Arabidopsis genome for genes involved in the regulation of mitochondrial DNA metabolism revealed a region of chromosome III that is unusually rich in genes for mitochondrial DNA and RNA maintenance. An apparently similar genetic linkage was observed in the rice genome. Several of the genes identified within the chromosome III interval appear to target the plastid or to be targeted dually to the mitochondria and the plastid, suggesting that the process of endosymbiosis likely is accompanied by an intimate coevolution of these two organelles for their genome maintenance functions.

Substoichiometric shifting in the plant mitochondrial genome is influenced by a gene homologous to MutS.

The plant mitochondrial genome is retained in a multipartite structure that arises by a process of repeat-mediated homologous recombination. Low-frequency ectopic recombination also occurs, often producing sequence chimeras, aberrant ORFs, and novel subgenomic DNA molecules. This genomic plasticity may distinguish the plant mitochondrion from mammalian and fungal types. In plants, relative copy number of recombination-derived subgenomic DNA molecules within mitochondria is controlled by nuclear genes, and a genomic shifting process can result in their differential copy number suppression to nearly undetectable levels. We have cloned a nuclear gene that regulates mitochondrial substoichiometric shifting in Arabidopsis. The CHM gene was shown to encode a protein related to the MutS protein of Escherichia coli that is involved in mismatch repair and DNA recombination. We postulate that the process of substoichiometric shifting in plants may be a consequence of ectopic recombination suppression or replication stalling at ectopic recombination sites to effect molecule-specific copy number modulation.