KNOX Genes

From Purdue Genomics Database Facility

KNOX (KNOTTED1-LIKE HOMEOBOX) Genes in Selaginella

Elizabeth Barker (barker.e@gmail.com) and Neil Ashton (physcomitrella@gmail.com)

Many important aspects of plant morphogenesis, including evolution and specification of the architecture of both vegetative and generative parts, are controlled by members of multigene families that encode transcription factors. Arguably the most significant of these are the MADS-box and KNOX gene families. Selaginella moellendorffii is currently the only lycophyte or, indeed, vascular cryptogam whose genome has been fully sequenced. Examination of its genome revealed 4 KNOX genes, a number that is comparable to that in Physcomitrella, which has 5 [1,8], 4 times the number in green algae [10, 11, 12] and half the number in Arabidopsis [4,7](Fig.1).

Figure 1. Comparison of the numbers of class 1 and 2 KNOX genes in the fully sequenced genomes of Ostreooccus lucimarinus [11], O. tauri [12], Chlamydomonas reinhardtii [4, 10], Physcomitrella patens [1, 8], Selaginella moellendorffii and Arabidopsis thaliana [4, 7].

Based on gene architecture and sequence, Selaginella has two class 1 and two class 2 KNOX genes consistent with the observation that in Physcomitrella and Arabidopsis half or approximately half of their KNOX genes comprise each subcategory [1, 4, 7] (Fig. 1, Table 1). The heterogeneous algal KNOX genes are characterised by sequences and gene models that differ from those of embryophytic KNOX genes to such an extent that they cannot be classified as class 1 or class 2.

Gene	Class	Protein ID and Scaffold Address		Exons	EST evidence?
KN1	1	KN1-1 450995 26:1418397-1420744	KN1-2 451276 34:1598451-1600796	5	Yes
KN2	1	KN2-1 159366 87:646301-649371	KN2-2 451297 119:122026-124845	6	Yes
KN3	2	KN3-1 451277 11:2439776-2441812	KN3-2 451279 23:1402714-1405042	5	Yes
KN4	2	KN4-1 450988 140:238475-239917	KN4-2 451281 164:184782-186352	5	Yes

Table 1. List of KNOX genes discovered in the genome of Selaginella moellendorffii, including both haplotypes of each gene are included. A fifth predicted gene, KN5, contains portions of the conserved KNOX gene sequence. One of its haplotypes contains a stop codon within conserved sequence. This sequence requires further analysis and, although EST evidence exists, may prove to be a pseudogene. Consequently, it was omitted from phylogenetic analyses and was not included in the histogram of gene numbers (Fig. 1).

Phylogenetic reconstructions (Fig. 2) demonstrate that Selaginella, Physcomitrella and Arabidopsis KNOX genes cluster in two, strongly supported, monophyletic clades corresponding exactly to the class 1 and 2 designations described above. These clades together comprise an embryophyte clade that excludes the unclassified algal sequences.

Figure 2. Unrooted phylogenetic tree of KNOX genes constructed by Bayesian analysis [6] of an alignment of KNOX protein amino acid sequences of the corresponding genes. Displayed numerical values indicate statistical support for each clade (posterior probabilites from Bayesian analysis above bootstrap percentages from weighted maximum parsimony [9]). Selaginella genes are bolded.

Unlike the MADS-box genes, KNOX genes have undergone a modest expansion of their numbers during evolution of the major terrestrial plant taxa with the most significant increase probably occurring in the lineage leading from algal chlorophytes to a common progenitor of embryophytes (Fig. 1). This observation together with recent studies of KNOX gene function in Selaginella [5] and Physcomitrella [8] implies that expansion and diversification into class 1 and 2 genes were required for evolution of the terrestrial plant body and successful colonisation of land. In vascular plants including lycophytes, class 1 genes are implicated in maintenance of the indeterminate SAM, internode development, development of compound leaves, positioning of megaphyllous and Selaginella microphyllous leaves, and bifurcation of Selaginella shoots [2, 4, 5, 7]. It has been proposed that the latter three roles require antagonistic interactions with other genes, in particular, ARP genes [2, 4, 5, 7]. The Physcomitrella genome does not contain an ARP homologue [3] and the moss sporophyte is both unbranched and determinate. Thus a more fundamental ancestral role for KNOX genes must be sought. One, which is consistent with recent findings in Physcomitrella, is the prevention of premature differentiation. Thus, we suggest that the primary function of class 1 KNOX genes during sporophytic development in early terrestrial plants was to prevent premature sporogenesis, thereby ensuring the production of a larger mass of sporophytic tissues, including sporogenous cells, prior to meiotic sporogenesis and consequently greater yields of genetically diverse spores. This would have facilitated spore dispersal and enhanced the efficiency of colonisation of terrestrial habitats [8]. The functions of class 2 genes are poorly understood although recent evidence suggests a role in the production of desiccation tolerant spore walls [8].

References

[1] C. E. M. Champagne, N. W. Ashton, New Phytol. 150, 23-36 (2001).

[2] C. Champagne, N. Sinha, Development, 131, 4401-4412 (2004).

[3] S. K. Floyd, J. L. Bowman, J. Plant Sci. 168, 1-35 (2007).

[4] S. Hake et al., Annu. Rev. Cell Biol. 20', 125-51 (2004).

[5] C. J. Harrison et al. Nature 434, 509-514 (2005).

[6] J. P. Huelsenbeck, F. Ronquist, Bioinformatics 17', 754-755 (2001).

[7] S. Scofield, J. A. H. Murray. Plant Mol. Biol. 60, 929-946 (2006).

[8] S. D. Singer, N. W. Ashton, Plant Cell Rep. 26', 2039-2054 (2007).

[9] D. L. Swofford, PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). 'Version 4 (Sinauer Associates, Sunderland, MA, 1998).

[10] U. S. Department of Energy, Joint Genome Institute, Chlamydomonas reinhardtii v.3.0, 2007; http://genome.jgi-psf.org/Chlre3/Chlre3.home.html

[11] U. S. Department of Energy, Joint Genome Institute, Ostreococcus lucimarinus v.2.0, 2006; http://genome.jgi-psf.org/Ost9901_3/Ost9901_3.home.html

[12] U. S. Department of Energy, Joint Genome Institute, Ostreococcus tauri v.2.0, 2006; http://genome.jgi-psf.org/Ostta4/Ostta4.home.html