A tale of two tissues: AtGH9C1 is an endo-ß-1,4-glucanase involved in root hair and endosperm development in Arabidopsis.

Arabidopsis AtGH9C1 is an endo-ß-1,4-glucanase possessing a carbohydrate-binding domain (CBM49). Analysis of AtGH9C1 expression by promoter-reporter GUS, RT-PCR, public transcriptome databases and GFP protein tagging demonstrated a high and selective expression of AtGH9C1 in root hairs and in the endosperm. Expression in root hair cells started prior to bulge formation and continued during hair elongation. AtGH9C1 expression increased with treatments that increase density (ACC) or length (sucrose) of root hairs. Expression in the endosperm extended sequentially to the micropylar, peripheral and chalazal compartments. A mutant with reduced AtGH9C1 expression had a delay in germination and a marked reduction in root hair presence. Complementation of the mutant partially improved both germination and root hair density. Experiments with ectopically expressed AtGH9C1-GFP with and without the CBM49, demonstrated that both forms of the protein are secreted and that CBM49 targets the protein to specific regions of the cell wall, but what makes these regions special is still unknown. The amino acid alignment of angiosperm GH9 genes with C-terminal extensions illustrate that AtGH9C1 belongs to a different clade than its tomato homolog, S1GH9C1. The latter has a CBM49 that was shown to bind crystalline cellulose. We suggest that AtGH9C1 is associated with the weakening of the cell wall during formation and growth of the root hair as well as with the sequential anterior-posterior breakdown of the endosperm cell wall that provides space for the growing embryo. Thus, is likely that the CBM49 of AtGH9C1 recognizes a form of cellulose or glucan polymer that is prevalent in the wall of these specialized tissues and that is different than the one recognized by S1GH9C1.

KORRIGAN1 and its aspen homolog PttCel9A1 decrease cellulose crystallinity in Arabidopsis stems.

KORRIGAN1 (KOR1) is a membrane-bound cellulase implicated in cellulose biosynthesis. PttCel9A1 from hybrid aspen (Populus tremula L. x tremuloides Michx.) has high sequence similarity to KOR1 and we demonstrate here that it complements kor1-1 mutants, indicating that it is a KOR1 ortholog. We investigated the function of PttCel9A1/KOR1 in Arabidopsis secondary growth using transgenic lines expressing 35S::PttCel9A1 and the KOR1 mutant line irx2-2. The presence of elevated levels of PttCel9A1/KOR1 in secondary walls of 35S::PttCel9A1 lines was confirmed by in muro visualization of cellulase activity. Compared with the wild type, 35S::PttCel9A1 lines had higher trifluoroacetic acid (TFA)-hydrolyzable glucan contents, similar Updegraff cellulose contents and lower cellulose crystallinity indices, as determined by (13)C solid-state nuclear magnetic resonance (NMR) spectroscopy. irx2-2 mutants had wild-type TFA-hydrolyzable glucan contents, but reduced Updegraff cellulose contents and higher than wild-type cellulose crystallinity indices. The data support the hypothesis that PttCel9A1/KOR1 activity is present in cell walls, where it facilitates cellulose biosynthesis in a way that increases the amount of non-crystalline cellulose.

Root cap specific expression of an endo-beta-1,4-D-glucanase (cellulase): a new marker to study root development in Arabidopsis.

The sloughing of root cap cells from the root tip is important because it assists the growing root in penetrating the soil. Using a promoter-reporter (GUS) and RT-PCR analysis, we identified an endo-beta-1,4-glucanase (AtCel5) of Arabidopsis thaliana that is expressed exclusively in root cap cells of both primary and secondary roots. Expression is inhibited by high concentrations of IAA, both exogenous and internal, as well as by ABA. AtCel5 expression begins once the mature tissue pattern is established and continues for 3 weeks. GUS staining is observed in both root cap cells that are still attached and cells that have already been shed. Using AtCel5-GUS as a marker, we observed that the root cap cells begin to separate at the sides of the tip while the cells of the central region of the tip separate last. Separation involves sequential tiers of intact cells that separate from the periphery of the root tip. A homozygous T-DNA insertion mutant that does not express AtCel5 forms the root cap and sheds root cap cells but sloughing is less efficient compared to wild type. The reduction in sloughing in the mutant does not affect the overall growth performance of the plant in loose media. The modest effect of abolishing AtCel5 expression suggests that there are multiple redundant genes regulating the process of sloughing of the root cap, including AtCel3/At1g71380, the paralog of the AtCel5 gene that is also expressed in the root cap cells. Thus, these two endo-1,4-beta-D-glucanases may have a role in the sloughing of border cells from the root tip. We propose that AtCel5, provides a new molecular marker to further analyze the process of root cap cell separation and a root cap specific promoter for targeting to the environment genes with beneficial properties for plant growth.