The only Class II KNOX gene that has recently been well characterized and extensively studied is KNAT7 [12,13,14,15,16,30,31]. The role of KNAT7 TF as a regulator in SCW biosynthesis was first reported in Arabidopsis through the observation of the irx (irregular xylem) phenotype that occurred in a loss-of-function knat7 mutant, irx11 [30]. At the same time, the tight co-expression of the KNAT7 TF gene along with SCW-specific CesA genes was reported using microarrays of Arabidopsis inflorescence stems undergoing SCW formation [11,32]. Promoter-GUS expression studies of AtKNAT7 in Arabidopsis showed that it is highly expressed in developing xylem, phloem fibers, and cambium cells of inflorescence stems [13]. Wang et al. [15] recently examined whether several Class II KNOX genes from Arabidopsis, KNAT3, KNAT4, KNAT5, and KNAT7, were expressed during SCW deposition. All these Class II KNOX gene promoters regulated GUS expression in the vascular bundles in younger stems and intrafascicular fibers and vessel cells in older stems. These observations suggest that these Class II KNOX genes have similar expression patterns during the deposition of the SCWs. Qin et al. [16] also showed that while KNAT7 expression was much higher in stem tissues, KNAT3 expression remained similar in all tissues examined. Promoter-GUS fusions confirmed that KNAT3 and KNAT7 genes are co-expressed in developing xylem and interfascicular fibers in the Arabidopsis stem.

In poplar (Populus balsamifera), the expression of PtKNAT7 gradually increases from the primary cell wall expansion stage to the mature xylem tissue formation stage, and from the youngest to the older internodes of stem [13]. Cotton GhKNL1 was reported to be preferentially expressed in developing cotton fibers during SCW biosynthesis [33]. Switchgrass KNAT7 also appears to be a functional ortholog of Arabidopsis KNAT7, based on its expression patterns [34]. In our laboratory, we studied the expression patterns of two Class II KNOX genes, KNAT3 and KNAT7, in tobacco (Nicotiana benthamiana) [14]. Higher expression of NbKNAT7 was seen in older stems of tobacco showing secondary growth followed by young stems and old leaves, while NbKNAT3 displayed higher expression in older leaves followed by roots and young leaves. These two Class II KNOX genes were also found to be highly expressed during tension wood formation in aspen. The expression of NbKNAT3 and NbKNAT7 in young and old stems indicates that they play a role in wood formation. Thus, Class II KNOX genes are associated with SCW formation during xylem and fiber development.

Until 2005, KNAT7 was not often discussed in mutation studies of the Class II KNOX genes; however, a number of Class II KNOX mutations have recently been studied in detail (Table 1). A T-DNA insertion in the intron of the KNAT7 gene resulted in a loss-of-function mutant, irx11, that showed only a moderately weak growth phenotype. The irx11 mutant also exhibited the typical irx phenotype in xylem vessels that were collapsed due to weak SCW formation. The irx11 mutant did not have significantly altered cellulose or xylan content compared to controls. No lignin content of these mutants was reported at that time. While discovering a set of novel TFs involved in SCW biosynthesis, Zhong et al. [12] associated KNAT7 expression with SCW formation, and the dominant repression of KNAT7 (DR-KNAT7 mutants) affected SCW formation in both xylem and fiber cells (Table 1). Curiously, they did not observe the typical irx phenomenon in these DR-KNAT7 mutants, a tell-tale sign of weak SCW formation; however, the cell wall thicknesses of both xylem vessels and fibers were reduced compared to controls (28% down in interfascicular fibers (IF), 26% down in vessels (V), and 80% down in xylary fibers (XF)). Several monosaccharides from the cell walls of DR-KNAT7 mutants were reduced by 20–30%, except for arabinose, which was increased by 18%. The overexpression of KNAT7 did not increase the SCW thickness of fibers and vessels. These results indicated that KNAT7 could be a positive regulator of SCW formation in Arabidopsis. However, Li et al. [13] reported a contrasting observation that loss-of-function mutants in the AtKNAT7 gene resulted in differential thicknesses of interfascicular and xylary fibers compared to vessels (58% up in IF, 35% down in V, and 31% up in XF; Table 1). The vessels walls were thinner, resulting in collapsed xylem vessels that showed the irx phenotype (similar to [30]); however, the interfascicular fibers were significantly thicker than in the wild type control, suggesting that KNAT7 is a transcriptional repressor of fiber SCW formation (but a transcriptional activator of vessel SCW formation). KNAT7 overexpression lines exhibited thinner fiber walls (57% down in IF) with normal vessel and xylary fiber cell walls. Interestingly, even though many SCW-specific cellulose and xylan synthesis genes were upregulated in these mutants, no quantitative changes in cellulose or xylan were reported. All ten lignin synthesis genes tested were upregulated along with an 11% increase in lignin content of cell walls from the stem. Li et al. [30] speculated that KNAT7 interacts with different partner proteins in different cell types to form functionally distinct complexes. Recently, the regulatory roles of other members of the Class II KNOX gene family, KNAT3, KNAT4, and KNAT5, in SCW formation were explored in Arabidopsis inflorescence stems [15,16] (Table 1). Loss-of-function mutants of knat3, knat4, and knat5 did not produce any irx phenotype, as observed in the case of loss-of-function mutants of knat7 [15]. This could be due to the functional redundancy of KNOX II genes. However, knat3/knat7 double mutants displayed an enhanced irx phenotype compared to single knat7 mutants. These double mutants had thinner interfascicular fiber cell walls compared to the single mutants and wild-type plants (40% down in IF) indicating a potentially positive regulatory role of KNAT3 in combination with KNAT7 in xylem SCW development. Even though many SCW genes were highly expressed in the knat3/knat7 double mutants, the cellulose and xylan contents of their cell walls were reduced by 19% and 43%, respectively, and the changes in lignin content were not significant. The Syringyl to Guaicyl (S/G) lignin ratio was down by 83%; however, it was not possible to correlate all these cell wall content changes with the changes in gene expression patterns. In addition, the severe irx phenotype in these double mutants indicated the overlapping roles and partial functional redundancy of KNAT3 and KNAT7 in xylem vessel development during SCW formation. Furthermore, KNAT3 overexpression in Arabidopsis resulted in thickened interfascicular fibers in the SCW of inflorescence stems [15]. This study described KNAT3 as a potential transcriptional activator, working together with KNAT7 to promote SCW biosynthesis in xylem vessels. A synergistic interaction of KNAT3 and KNAT7 to regulate monolignol biosynthesis in Arabidopsis was also reported in another study [16]. Most importantly, they attempted to link S-lignin formation with KNAT3 and KNAT7 expression; however, they could not show the direct transcriptional regulation of a key gene, ferulate 5-hydroxylase (F5H), involved in S-lignin formation by KNAT3 or KNAT7. Similar to the earlier observation by Wang et al. [15], the overexpression of KNAT3 also caused thickening in the interfascicular fiber walls, indicating the positive regulation of interfascicular fiber wall development by KNAT3. These studies by Wang et al. and Qin et al. [15,16] reconciled the paradoxical observations about KNAT7 mutants in Arabidopsis and indicated that KNAT3 and KNAT7 might be working synergistically in fibers, but antagonistically in vessels, during the regulation of SCW biosynthesis (Table 1).