Boundary genes in regulation and evolution of secondary growth

Abstract

Many extant land plants display secondary growth originating in a lateral meristem known as vascular cambium. A conspicuous product of secondary growth is wood which dominates terrestrial ecosystem biomass. Despite the economic and ecological significance of the process the underlying molecular mechanism are still poorly understood. We have recently shown that members of the LBD transcription factor family play function in control of secondary growth. Here we propose a mechanistic model of LBD regulatory roles. We also show how these roles may be linked to evolutionary changes in level and pattern of wood formation that provide structural and functional innovations in wood anatomy in relation to species growth habit and biology.

Secondary growth originating in a lateral meristem known as vascular cambium is responsible for stem girth expansion which in addition to providing mechanical support to the growing stem has important functions in transport, storage and defense against pathogens. Because of the importance of these processes to survival, there is a significant environmental and taxonomic variation in the level, organization and patterns of vascular cambium outgrowth which are tightly linked to essential adaptations. Very little is known about the mechanisms that regulate the process and have played role in its evolutionary change.¹

Although many land plants have a vascular cambium, only in trees it displays exaggerated and perennial outgrowth that contributes substantially to the trees' total biomass. In most extant trees, vascular cambium consists of meristem cells (cambial initials) organized in radial files that form continuous cylinder around the stem. Cambium initials divide periclinally to produce xylem, phloem and ray mother cells.^2–4 Mother cells then undergo several rounds of division resulting in a layer of poorly differentiated and morphologically indistinguishable cells that is known as the cambium zone. The cambium zone is a boundary between the cambium and the flanking differentiated xylem and phloem. In this zone cells gradually loose pluripotent meristem identity and acquire a specific cell fate to become vessels, fibers, sieve elements and rays.^4,5 We have recently shown⁶ that this boundary region is likely under similar regulation as the one described in SAM which separates the stem cell niche from the emerging lateral organ primordia. An uncharacterized member of the LATERAL ORGAN BOUNDARIES (LBD) transcription factor family⁷ from poplar (PtLBD1) was found to regulate secondary phloem and rays development.⁶ The gene was expressed on the phloem side of the cambium zone and into the differentiating secondary phloem. PtLBD1 transgenic upregulation or dominant negative suppression caused increased or decreased secondary phloem production respectively. Expression analysis of putative gene targets suggested that this function is likely and similarly as in SAM mediated through suppression of genes that promote meristem cell identity (e.g., KNOXI, class I KNOTTED-LIKE HOMEOBOX) and activation of genes that trigger differentiation of phloem (e.g., APL, ALTERED PHLOEM DEVELOPMENT). Expression survey of the whole poplar LBD family (57 members) indicates that two LBD genes (PtLBD1 and PtLBD4) are specifically expressed in secondary phloem and two in secondary xylem (PtLBD18 and PtLBD30). This led us to propose a model of LBD role in secondary woody growth (Fig. 1). According to the model, LBD1 and LBD4 are expressed at the cambium/phloem boundary and play role in regulation of secondary phloem development through restricting the expression of meristem identity genes like KNOXI in the cambium zone^8,9 and promoting phloem development through activation of genes like APL¹⁰ and likely others. LBD18 and LBD30 are expressed at the cambium/xylem border and also restrain the expression of meristem identity genes to the cambium zone while at the same time promote xylem development through activation of unknown set of genes.

Figure 1.

Hypothetical model of LBD function during secondary woody growth. According to the model, LBDs play antagonistic roles with meristem maintenance genes in order to maintain meristem identity in cambium and/or promote xylem/phloem/ray cell tissue differentiation. The dark to light gradient represents loss of meristem cell identity and differentiation.

The apparent essential regulatory roles of members of LBD gene family in regulation of wood formation suggest that they may have played significant role in evolution of woody growth habit as well as different wood anatomies. We therefore believe that changes in the number (and/or expression) of these genes through plant evolution may have provided structural and functional evolutionary innovations in wood anatomy in relation to species growth habit and biology. We have taken advantage of the large number of sequenced plant genomes to investigate the orthologous relationships of the poplar four genes with these from other plant species (Table 1). We found that LBD proteins are present in all plants. We could not identify LBDs in algae suggesting that the whole gene family has evolved after the colonization of land plants. A large and highly significant expansion of PtLBD1 lineage was found in Vitis vinifera genome. There were 13 putative LBD1 orthologs (Fig. 2B, Table 1). Vitis is vine and has a unique wood anatomy^11,12 (Fig. 2A). Most notably, the rays are highly multiseriate. The intercalation of less-lignified tissues (like rays) in the xylem of vines and lianas is an adaptive feature allowing more stem flexibility.¹ In addition, the secondary phloem is well-developed with multiple growth rings. The multiseriate rays and increased secondary phloem production in Vitis resembles the poplar transgenics with increased PtLBD1 expression (Fig. 2A). Thus the putative increased LBD1 gene dosage in Vitis corresponds to its wood anatomical features.

Table 1.

Orthologs of 4 poplar LBDs involved in regulation of secondary growth

	Species*
Genes	Ptr	Vvi	Ath	Aco	Osa	Smo	Ppa
LBD1	3	13	2	1	1	0	0
LBD4	4	3	1	1	1	2	3
LBD15	2	2	1	1	1	1	2
LBD18	5	1	2	2	2	0	0

^*Species abbreviation: Ptr-Populus trichocarpa; Vvi-Vitis vinifera; Ath-Arabidopsis thaliana; Aco-Aquilegia caerulea; Osa-Oryza sativa; Smo-Selaginella moellendorffii; Ppa-Physcomitrella patens.

Figure 2.

Expansion of LBD1 lineage in Vitis correlates with multiseriate rays' formation. (A) Wood anatomy in wild type (WT) Vitis plants, WT poplar (Populus tremula X Populus alba) P35S:PtaLBD1 poplar transgenic plants generated in the same Populus tremula X Populus alba genotype. (B) Phylogenetic tree of LBD1 members from Arabidopsis thaliana, Populus trichocarpa and Vitis vinifera. Numbers in branches indicate bootrstrap support of 1,000 iterations. Arabidopsis Genome Identity (AGI) number (starts with AT), Populus gene identifier (starts with POPTR) and Vitis models numbers (starts with GSVIVG) are according phytozome.org. The underlined Populus model corresponds to PtaLBD1 gene as previously described in reference ⁶.

Our studies provide new inroads into understanding the regulation and evolution of secondary growth.

Addendum to: Yordanov YS, Regan S, Busov V. Members of the Lateral Organ Boundaries Domain (LBD) transcription factors family are involved in regulation of secondary growth in Populus. Plant Cell. 2010;22:3662–3677. doi: 10.1105/tpc.110.078634.