A new cellulose synthase gene (PtrCesA2) from aspen xylem is orthologous to Arabidopsis AtCesA7 (irx3) gene associated with secondary cell wall synthesis

We report here the molecular cloning and characterization of a new full-length cellulose synthase (CesA) cDNA, PtrCesA2 from aspen (Populus tremuloides) trees. The predicted PtrCesA2 protein shows a high degree of identity/similarity (87%/91%) to the predicted gene product of Arabidopsis AtCesA7 gene that has been associated with secondary cell wall development. Previously, a mutation in AtCesA7 gene (irx3) was correlated with a significant decrease in the amount of cellulose synthesized (about 70%) and genetic complementation of irx3 mutant with a wild-type AtCesA7 gene restored the normal phenotype. This is the first report of a full-length AtCesA7 ortholog from any non-Arabidopsis species. Interestingly, PtrCesA2 shares only 64% identity with our earlier reported PtrCesA1 from aspen suggesting its structural distinctness from the only other known CesA member from the aspen genome. PtrCesA1 is a xylem-specific and tension stress responsive gene that is highly similar to another Arabidopsis gene, AtCesA8 which also has been associated with secondary wall development. Moreover, AtCesA7 and AtCesA8 are suggested to be part of the same cellulose synthase complex. Isolation of PtrCesA2 from a xylem library enriched in cells with active secondary wall synthesis, PtrCesA2 expression levels similar to PtrCesA1 and high similarity of PtrCesA1 and PtrCesA2 to AtCesA8 and AtCesA7, respectively, suggest that both these aspen genes might be involved in the secondary wall development in aspen woody tissues. Availability of two aspen CesA orthologs will now enable us to examine if PtrCesA1 and PtrCesA2 functionally interact during aspen wood development that has long-term implications on genetic improvement of forest trees.     

The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana

The irregular xylem 2 (irx2) mutant of Arabidopsis thaliana exhibits a cellulose deficiency in the secondary cell wall, which is brought about by a point mutation in the KORRIGAN (KOR) beta,1-4 endoglucanase (beta,1-4 EGase) gene. Measurement of the total crystalline cellulose in the inflorescence stem indicates that the irx2 mutant contains approximately 30% of the level present in the wild type (WT). Fourier-Transform Infra Red (FTIR) analysis, however, indicates that there is no decrease in cellulose in primary cell walls of the cortical and epidermal cells of the stem. KOR expression is correlated with cellulose synthesis and is highly expressed in cells synthesising a secondary cell wall. Co-precipitation experiments, using either an epitope-tagged form of KOR or IRX3 (AtCesA7), suggest that KOR is not an integral part of the cellulose synthase complex. These data are supported by immunolocalisation of KOR that suggests that KOR does not localise to sites of secondary cell wall deposition in the developing xylem. The defect in irx2 plant is consistent with a role for KOR in the later stages of secondary cell wall formation, suggesting a role in processing of the growing microfibrils or release of the cellulose synthase complex.     

Structure of cellulose-deficient secondary cell walls from the irx3 mutant of Arabidopsis thaliana

In the Arabidopsis mutant irx3, truncation of the AtCesA7 gene encoding a xylem-specific cellulose synthase results in reduced cellulose synthesis in the affected xylem cells and collapse of mature xylem vessels. Here we describe spectroscopic experiments to determine whether any cellulose, normal or abnormal, remained in the walls of these cells and whether there were consequent effects on other cell-wall polysaccharides. Xylem cell walls from irx3 and its wild-type were prepared by anatomically specific isolation and were examined by solid-state NMR spectroscopy and FTIR microscopy. The affected cell walls of irx3 contained low levels of crystalline cellulose, probably associated with primary cell walls. There was no evidence that crystalline cellulose was replaced by less ordered glucans. From the molecular mobility of xylans and lignin it was deduced that these non-cellulosic polymers were cross-linked together in both irx3 and the wild-type. The disorder previously observed in the spatial pattern of non-cellulosic polymer deposition in the secondary walls of irx3 xylem could not be explained by any alteration in the structure or cross-linking of these polymers and may be attributed directly to the absence of cellulose microfibrils which, in the wild-type, scaffold the organisation of the other polymers into a coherent secondary cell wall.     

The irregular xylem3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis.

The irregular xylem3 (irx3) mutant of Arabidopsis has a severe deficiency in secondary cell wall cellulose deposition that leads to collapsed xylem cells. The irx3 mutation has been mapped to the top arm of chromosome V near the marker nga106. Expressed sequence tag clone 75G11, which exhibits sequence similarity to cellulose synthase, was found to be tightly linked to irx3, and genomic clones containing the gene corresponding to clone 75G11 complemented the irx3 mutation. Thus, the IRX3 gene encodes a cellulose synthase component that is specifically required for the synthesis of cellulose in the secondary cell wall. The irx3 mutant allele contains a stop codon that truncates the gene product by 168 amino acids, suggesting that this allele is null. Furthermore, in contrast to radial swelling1 (rsw1) plants, irx3 plants show no increase in the accumulation of beta-1,4-linked glucose in the noncrystalline cell wall fraction. IRX3 and RSW1 fall into a distinct subgroup (Csa) of Arabidopsis genes showing homology to bacterial cellulose synthases.     

The Arabidopsis calcium-dependent protein kinase, CPK6, functions as a positive regulator of methyl jasmonate signaling in guard cells

Previous studies have demonstrated that methyl jasmonate (MeJA) induces stomatal closure dependent on change of cytosolic free calcium concentration in guard cells. However, these molecular mechanisms of intracellular Ca(2+) signal perception remain unknown. Calcium-dependent protein kinases (CDPKs) function as Ca(2+) signal transducers in various plant physiological processes. It has been reported that four Arabidopsis (Arabidopsis thaliana) CDPKs, CPK3, CPK6, CPK4, and CPK11, are involved in abscisic acid signaling in guard cells. It is also known that there is an interaction between MeJA and abscisic acid signaling in guard cells. In this study, we examined the roles of these CDPKs in MeJA signaling in guard cells using Arabidopsis mutants disrupted in the CDPK genes. Disruption of the CPK6 gene impaired MeJA-induced stomatal closure, but disruption of the other CDPK genes did not. Despite the broad expression pattern of CPK6, we did not find other remarkable MeJA-insensitive phenotypes in the cpk6-1 mutant. The whole-cell patch-clamp analysis revealed that MeJA activation of nonselective Ca(2+)-permeable cation channels is impaired in the cpk6-1 mutant. Consistent with this result, MeJA-induced transient cytosolic free calcium concentration increments were reduced in the cpk6-1 mutant. MeJA failed to activate slow-type anion channels in the cpk6-1 guard cells. Production of early signal components, reactive oxygen species and nitric oxide, in guard cells was elicited by MeJA in the cpk6-1 mutant as in the wild type. These results provide genetic evidence that CPK6 has a different role from CPK3 and functions as a positive regulator of MeJA signaling in Arabidopsis guard cells.     

FLOWERING LOCUS T regulates stomatal opening

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange for photosynthesis in response to light, CO(2), and phytohormone abscisic acid. Phototropins (phot1 and phot2) are plant blue-light receptor kinases and mediate stomatal opening via activation of the plasma membrane H(+)-ATPase. However, the signaling mechanism from phototropins to the H(+)-ATPase has yet to be determined. Here, we show that FLOWERING LOCUS T (FT) is expressed in guard cells and regulates stomatal opening. We isolated an scs (suppressor of closed-stomata phenotype in phot1 phot2) 1-1 mutant of Arabidopsis thaliana and showed that scs1-1 carries a novel null early flowering 3 (elf3) allele in a phot1 phot2 background. scs1-1 (elf3 phot1 phot2 triple mutant) had an open-stomata phenotype with high H(+)-ATPase activity and showed increased levels of FT mRNA in guard cells. Transgenic plants overexpressing FT in guard cells showed open stomata, whereas a loss-of-function FT allele, ft-1, exhibited closed stomata and failed to activate the H(+)-ATPase in response to blue light. Our results define a new cell-autonomous role for FT and demonstrate that the flowering time genes ELF3 and FT are involved in the regulation of H(+)-ATPase by blue light in guard cells.     

Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis

Previous studies using co-expression analysis have identified a large number of genes likely to be involved in secondary cell-wall formation. However, the function of very few of these genes is known. We have studied the cell-wall phenotype of irx7, irx8 and irx9, three previously described irregular xylem (irx) mutants, and irx14 and parvus-3, which we now show also to be secondary cell-wall mutants. All five mutants, which have mutations in genes encoding putative glycosyltransferases, exhibited large decreases in xylan. In addition, all five mutants were found to have the same specific defect in xylan structure, retaining MeGlcUA but lacking GlcUA side branches. Polysaccharide analysis by carbohydrate gel electrophoresis (PACE) was used to determine the xylan structure in Arabidopsis, and revealed that side branches are added to approximately one in every eight xylose residues. Interestingly, this ratio is constant in all the lines analysed despite the wide variation in xylan content and the absence of GlcUA branches. Xylanase digestion of xylan from wild-type plants released a short oligosaccharide sequence at the reducing end of the xylan chain. MALDI-TOF MS analysis indicated that this sequence of sugars was absent in xylan from irx7, irx8 and parvus-3 mutants, but was present in irx9 and irx14. This is consistent with previous NMR analysis of xylan from irx7, irx8 and irx9, and suggests that PARVUS may be involved in the synthesis of a xylan primer whereas IRX14 may be required to synthesize the xylan backbone. This hypothesis is supported by assays showing that irx9 and irx14 are both defective in incorporation of radiolabel from UDP (14)C-xylose. This study has important implications for both our understanding of xylan biosynthesis and the functional analysis of cell-wall biosynthesis genes.     

Characterization of IRX10 and IRX10-like reveals an essential role in glucuronoxylan biosynthesis in Arabidopsis

Xylan, the major hemicellulosic polysaccharide in Arabidopsis secondary cell walls, requires a number of glycosyltransferases (GT) to catalyse formation of the various glycosidic linkages found in the polymer. In this study, we characterized IRX10 and IRX10-like ( IRX10-L ), two highly homologous genes encoding members of the glycosyltransferase family 47 (GT47). T-DNA insertions in IRX10 gave a mild irregular xylem (irx) phenotype consistent with a minor defect in secondary cell-wall synthesis, whereas plants containing mutations in IRX10-L showed no change. However, irx10 irx10-L double mutant plants showed a much more severe irx and whole-plant phenotype, suggesting considerable functional redundancy between these two genes. Detailed biochemical analysis of the irx10 irx10-L double mutant showed a large reduction of xylan in the secondary cell walls, consistent with a specific defect in xylan biosynthesis. Furthermore, the irx10 irx10-L mutant retains the unique oligosaccharide found at the reducing end of Arabidopsis xylan, but shows a severe reduction in β(1,4) xylosyltransferase activity. These characteristics are similar to those of irx9 and irx14 , mutants that are believed to be defective in xylan chain elongation, and suggests that IRX10 and IRX10-L also play a role in elongation of the xylan backbone.     

Localization,ion channel regulation,and genetic interactions during abscisic acid signaling of the nuclear mRNA cap-binding protein,ABH1

Abscisic acid (ABA) regulates developmental processes and abiotic stress responses in plants. We recently characterized a new Arabidopsis mutant, abh1, which shows ABA-hypersensitive regulation of seed germination, stomatal closing, and cytosolic calcium increases in guard cells (V. Hugouvieux, J.M. Kwak, J.I. Schroeder [2001] Cell 106: 477-487). ABH1 encodes the large subunit of a dimeric Arabidopsis mRNA cap-binding complex and in expression profiling experiments was shown to affect mRNA levels of a subset of genes. Here, we show that the dimeric ABH1 and AtCBP20 subunits are ubiquitously expressed. Whole-plant growth phenotypes of abh1 are described and properties of ABH1 in guard cells are further analyzed. Complemented abh1 lines expressing a green fluorescent protein-ABH1 fusion protein demonstrate that ABH1 mainly localizes in guard cell nuclei. Stomatal apertures were smaller in abh1 compared with wild type (WT) when plants were grown at 40% humidity, and similar at 95% humidity. Correlated with stomatal apertures from plants grown at 40% humidity, slow anion channel currents were enhanced and inward potassium channel currents were decreased in abh1 guard cells compared with WT. Gas exchange measurements showed similar primary humidity responses in abh1 and WT, which together with results from abh1/abi1-1 double-mutant analyses suggest that abh1 shows enhanced sensitivity to endogenous ABA. Double-mutant analyses of the ABA-hypersensitive signaling mutants, era1-2 and abh1, showed complex genetic interactions, suggesting that ABH1 and ERA1 do not modulate the same negative regulator in ABA signaling. Mutations in the RNA-binding protein sad1 showed hypersensitive ABA-induced stomatal closing, whereas hyl1 did not affect this response. These data provide evidence for the model that the mRNA-processing proteins ABH1 and SAD1 function as negative regulators in guard cell ABA signaling.     

Functional analysis of complexes with mixed primary and secondary cellulose synthases

In higher plants, cellulose is synthesized by cellulose synthase complexes, which contain multiple isoforms of cellulose synthases (CESAs). Among the total 10 CESA genes in Arabidopsis, recessive mutations at three of them cause the collapse of mature xylem cells in inflorescence stems of Arabidopsis (irx1^cesa8, irx3^cesa7 and irx5^cesa4). These CESA genes are considered secondary cell wall CESAs. The others (the function CESA10 is still unknown) are thought to be specialized for cellulose synthesis in the primary cell wall. A split-ubiquitin membrane yeast two-hybrid system was used to assess interactions among four primary CESAs (CESA1, CESA2, CESA3, CESA6) and three secondary CESAs (CESA4, CESA7, CESA8). Our results showed that primary CESAs could physically interact with secondary CESAs in a limited fashion. Analysis of transgenic lines showed that CESA1 could partially rescue irx1^cesa8 null mutants, resulting in complementation of the plant growth defect, collapsed xylem and cellulose content deficiency. These results suggest that mixed primary and secondary CESA complexes are functional using experimental set-ups.     

Endogenous abscisic acid is involved in methyl jasmonate-induced reactive oxygen species and nitric oxide production but not in cytosolic alkalization in Arabidopsis guard cells

We recently demonstrated that endogenous abscisic acid (ABA) is involved in methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we investigated whether endogenous ABA is involved in MeJA-induced reactive oxygen species (ROS) and nitric oxide (NO) production and cytosolic alkalization in guard cells using an ABA-deficient Arabidopsis mutant, aba2-2, and an inhibitor of ABA biosynthesis, fluridon (FLU). The aba2-2 mutation impaired MeJA-induced ROS and NO production. FLU inhibited MeJA-induced ROS production in wild-type guard cells. Pretreatment with 0.1 μM ABA, which does not induce stomatal closure in the wild type, complemented the insensitivity to MeJA of the aba2-2 mutant. However, MeJA induced cytosolic alkalization in both wild-type and aba2-2 guard cells. These results suggest that endogenous ABA is involved in MeJA-induced ROS and NO production but not in MeJA-induced cytosolic alkalization in Arabidopsis guard cells.     

SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis

Ubiquitination plays important roles in plant hormone signal transduction. We show that the RING finger E3 ligase, Arabidopsis thaliana SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1), is involved in abscisic acid (ABA)-related stress signal transduction. SDIR1 is expressed in all tissues of Arabidopsis and is upregulated by drought and salt stress, but not by ABA. Plants expressing the ProSDIR1-beta-glucuronidase (GUS) reporter construct confirmed strong induction of GUS expression in stomatal guard cells and leaf mesophyll cells under drought stress. The green fluorescent protein-SDIR1 fusion protein is colocalized with intracellular membranes. We demonstrate that SDIR1 is an E3 ubiquitin ligase and that the RING finger conservation region is required for its activity. Overexpression of SDIR1 leads to ABA hypersensitivity and ABA-associated phenotypes, such as salt hypersensitivity in germination, enhanced ABA-induced stomatal closing, and enhanced drought tolerance. The expression levels of a number of key ABA and stress marker genes are altered both in SDIR1 overexpression and sdir1-1 mutant plants. Cross-complementation experiments showed that the ABA-INSENSITIVE5 (ABI5), ABRE BINDING FACTOR3 (ABF3), and ABF4 genes can rescue the ABA-insensitive phenotype of the sdir1-1 mutant, whereas SDIR1 could not rescue the abi5-1 mutant. This suggests that SDIR1 acts upstream of those basic leucine zipper family genes. Our results indicate that SDIR1 is a positive regulator of ABA signaling.     

Arabidopsis mutants of AtABCG22, an ABC transporter gene, increase water transpiration and drought susceptibility

In plants, water vapour is released into the atmosphere through stomata in a process called transpiration. Abscisic acid (ABA) is a key phytohormone that facilitates stomatal closure through its action on guard cells. Recently, ATP-binding cassette (ABC) transporter genes, AtABCG25 and AtABCG40, were shown to be involved in ABA transport and responses. However, the functions of many other AtABCG family genes are still unknown. Here, we identified another ABCG gene (AtABCG22) that is required for stomatal regulation in Arabidopsis. The atabcg22 mutant plants had lower leaf temperatures and increased water loss, implying elevated transpiration through an influence on stomatal regulation. We also found that atabcg22 plants were more suspectible to drought stress than wild-type plants. AtABCG22 was expressed in aerial organs, mainly guard cells, in which the gene expression pattern was consistent with the mutant phenotypes. Using double mutants, we investigated the genetic relationships between the mutations. The atabcg22 mutation further increased the water loss of srk2e/ost1 mutants, which were defective in ABA signalling in guard cells. Also, the atabcg22 mutation enhanced the phenotype of nced3 mutants, which were defective in ABA biosynthesis. Accordingly, the additive roles of AtABCG22 functions in ABA signalling and ABA biosynthesis are discussed.     

Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis

The irregular xylem 1 (irx1) mutant of Arabidopsis has a severe deficiency in the deposition of cellulose in secondary cell walls, which results in collapsed xylem cells. This mutation has been mapped to a 140-kb region of chromosome 4. A cellulose synthase catalytic subunit was found to be located in this region, and genomic clones containing this gene complemented the irx1 mutation. IRX1 shows homology to a previously described cellulose synthase (IRX3). Analysis of the irx1 and irx3 mutant phenotypes demonstrates that both IRX1 and IRX3 are essential for the production of cellulose in the same cell. Thus, IRX1 and IRX3 define distinct classes of catalytic subunits that are both essential for cellulose synthesis in plants. This finding is supported by coprecipitation of IRX1 with IRX3, suggesting that IRX1 and IRX3 are part of the same complex.     

Gene trap lines identify Arabidopsis genes expressed in stomatal guard cells

We employed a gene trap approach to identify genes expressed in stomatal guard cells of Arabidopsis thaliana . We examined patterns of reporter gene expression in approximately 20 000 gene trap lines, and recovered five lines with exclusive or preferential expression in stomata. The screen yielded two insertions in annotated genes, encoding the CYTOCHROME P450 86A2 (CYP86A2) mono-oxygenase, and the PLEIOTROPIC DRUG RESISTANCE 3 (AtPDR3) transporter. Expression of the trapped genes in guard cells was confirmed by RT-PCR experiments in purified stomata. Examination of homozygous mutant lines revealed that abscisic acid (ABA)-induced stomatal closure was impaired in the atpdr3 mutant. In three lines, insertions occurred outside transcribed units. Expression analysis of the genes surrounding the trapping inserts identified two genes selectively expressed in guard cells, corresponding to a PP2C PROTEIN PHOSPHATASE and an unknown expressed protein gene. Statistical analyses of the chromosomal regions tagged by the gene trap insertions revealed an over-represented [A/T]AAAG motif, previously described as an essential cis -active element for gene expression in stomata. The lines described in this work identify novel genes involved in the modulation of stomatal activity, provide useful markers for the study of developmental pathways in guard cells, and are a valuable source of guard cell-specific promoters.     

Arabidopsis genes IRREGULAR XYLEM (IRX15) and IRX15L encode DUF579-containing proteins that are essential for normal xylan deposition in the secondary cell wall

There are 10 genes in the Arabidopsis genome that contain a domain described in the Pfam database as domain of unknown function 579 (DUF579). Although DUF579 is widely distributed in eukaryotic species, there is no direct experimental evidence to assign a function to it. Five of the 10 Arabidopsis DUF579 family members are co‐expressed with marker genes for secondary cell wall formation. Plants in which two closely related members of the DUF579 family have been disrupted by T‐DNA insertions contain less xylose in the secondary cell wall as a result of decreased xylan content, and exhibit mildly distorted xylem vessels. Consequently we have named these genes IRREGULAR XYLEM 15 (IRX15) and IRX15L. These mutant plants exhibit many features of previously described xylan synthesis mutants, such as the replacement of glucuronic acid side chains with methylglucuronic acid side chains. By contrast, immunostaining of xylan and transmission electron microscopy (TEM) reveals that the walls of these irx15 irx15l double mutants are disorganized, compared with the wild type or other previously described xylan mutants, and exhibit dramatic increases in the quantity of sugar released in cell wall digestibility assays. Furthermore, localization studies using fluorescent fusion proteins label both the Golgi and also an unknown intracellular compartment. These data are consistent with irx15 and irx15l defining a new class of genes involved in xylan biosynthesis. How these genes function during xylan biosynthesis and deposition is discussed.