Hong Joo Cho

Disruption of Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Gene Altered Cuticular Lipid Composition, Increased Plastoglobules, and Enhanced Susceptibility to Infection by the Fungal Pathogen Alternaria brassicicola

All aerial parts of vascular plants are covered with cuticular waxes, which are synthesized by extensive export of intracellular lipids from epidermal cells to the surface. Although it has been suggested that plant lipid transfer proteins (LTPs) are involved in cuticular lipid transport, the in planta evidence is still not clear. In this study, a glycosylphosphatidylinositol-anchored LTP (LTPG1) showing higher expression in epidermal peels of stems than in stems was identified from an Arabidopsis (Arabidopsis thaliana) genome-wide microarray analysis. The expression of LTPG1 was observed in various tissues, including the epidermis, stem cortex, vascular bundles, mesophyll cells, root tips, pollen, and early-developing seeds. LTPG1 was found to be localized in the plasma membrane. Disruption of the LTPG1 gene caused alterations of cuticular lipid composition, but no significant changes on total wax and cutin monomer loads were seen. The largest reduction (10 mass %) in the ltpg1 mutant was observed in the C29 alkane, which is the major component of cuticular waxes in the stems and siliques. The reduced content was overcome by increases of the C29 secondary alcohols and C29 ketone wax loads. The ultrastructure analysis of ltpg1 showed a more diffuse cuticular layer structure, protrusions of the cytoplasm into the vacuole in the epidermis, and an increase of plastoglobules in the stem cortex and leaf mesophyll cells. Furthermore, the ltpg1 mutant was more susceptible to infection by the fungus Alternaria brassicicola than the wild type. Taken together, these results indicated that LTPG1 contributed either directly or indirectly to cuticular lipid accumulation.

Two Arabidopsis 3-ketoacyl CoA synthase genes, KCS20 and KCS2/DAISY, are functionally redundant in cuticular wax and root suberin biosynthesis, but differentially controlled by osmotic stress

Very-long-chain fatty acids (VLCFAs) are essential precursors of cuticular waxes and aliphatic suberins in roots. The first committed step in VLCFA biosynthesis is condensation of C(2) units to an acyl CoA by 3-ketoacyl CoA synthase (KCS). In this study, two KCS genes, KCS20 and KCS2/DAISY, that showed higher expression in stem epidermal peels than in stems were isolated. The relative expression of KCS20 and KCS2/DAISY transcripts was compared among various Arabidopsis organs or tissues and under various stress conditions, including osmotic stress. Although the cuticular waxes were not significantly altered in the kcs20 and kcs2/daisy-1 single mutants, the kcs20 kcs2/daisy-1 double mutant had a glossy green appearance due to a significant reduction of the amount of epicuticular wax crystals on the stems and siliques. Complete loss of KCS20 and KCS2/DAISY decreased the total wax content in stems and leaves by 20% and 15%, respectively, and an increase of 10-34% was observed in transgenic leaves that over-expressed KCS20 or KCS2/DAISY. The stem wax phenotype of the double mutant was rescued by expression of KSC20. In addition, the kcs20 kcs2/daisy-1 roots exhibited growth retardation and abnormal lamellation of the suberin layer in the endodermis. When compared with the single mutants, the roots of kcs20 kcs2/daisy-1 double mutantss exhibited significant reduction of C(22) and C(24) VLCFA derivatives but accumulation of C(20) VLCFA derivatives in aliphatic suberin. Taken together, these findings indicate that KCS20 and KCS2/DAISY are functionally redundant in the two-carbon elongation to C(22) VLCFA that is required for cuticular wax and root suberin biosynthesis. However, their expression is differentially controlled under osmotic stress conditions.

The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis

The tracheary elements (TEs) of the xylem serve as the water-conducting vessels of the plant vascular system. To achieve this, TEs undergo secondary cell wall thickening and cell death, during which the cell contents are completely removed. Cell death of TEs is a typical example of developmental programmed cell death that has been suggested to be autophagic. However, little evidence of autophagy in TE differentiation has been provided. The present study demonstrates that the small GTP binding protein RabG3b plays a role in TE differentiation through its function in autophagy. Differentiating wild type TE cells were found to undergo autophagy in an Arabidopsis culture system. Both autophagy and TE formation were significantly stimulated by overexpression of a constitutively active mutant (RabG3bCA), and were inhibited in transgenic plants overexpressing a dominant negative mutant (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid insensitive mutant bri1-301, and an autophagy mutant atg5-1. Taken together, our results suggest that autophagy occurs during TE differentiation, and that RabG3b, as a component of autophagy, regulates TE differentiation.

The Rab GTPase RabG3b Positively Regulates Autophagy and Immunity-Associated Hypersensitive Cell Death in Arabidopsis

A Rab GTPase protein connects autophagy with plant immunity-triggered hypersensitive response and programmed cell death. A central component of the plant defense response to pathogens is the hypersensitive response (HR), a form of programmed cell death (PCD). Rapid and localized induction of HR PCD ensures that pathogen invasion is prevented. Autophagy has been implicated in the regulation of HR cell death, but the functional relationship between autophagy and HR PCD and the regulation of these processes during the plant immune response remain controversial. Here, we show that a small GTP-binding protein, RabG3b, plays a positive role in autophagy and promotes HR cell death in response to avirulent bacterial pathogens in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing a constitutively active RabG3b (RabG3bCA) displayed accelerated, unrestricted HR PCD within 1 d of infection, in contrast to the autophagy-defective atg5-1 mutant, which gradually developed chlorotic cell death through uninfected sites over several days. Microscopic analyses showed the accumulation of autophagic structures during HR cell death in RabG3bCA cells. Our results suggest that RabG3b contributes to HR cell death via the activation of autophagy, which plays a positive role in plant immunity-triggered HR PCD.

Role of an Arabidopsis Rab GTPase RabG3b in Pathogen Response and Leaf Senescence

In our previous proteomic analysis, we isolated a small GTPase RabG3b as a salicylic acid-responsive protein in Arabidopsis (Oh et al. in Plant Cell 17:2832–2847, 2005). Here, we constructed transgenic plants overexpressing wild-type (RabG3bOX), constitutively active (RabG3bCA), and dominant negative (RabG3bDN) forms of RabG3b for functional studies. The phenotypes of these transgenic plants were indistinguishable from wild-type plants under normal growth conditions. However, both RabG3bOX and RabG3bCA plants displayed unrestricted hypersensitive programmed cell death against a fungal toxin Fumonisin B1 and a fungal pathogen Alternaria brassicicola, whereas no major difference between wild-type and RabG3bDN plants was observed. In addition, RabG3bOX and RabG3bCA plants underwent accelerated leaf senescence compared to wild-type and RabG3bDN plants. These results suggest that RabG3b is a modulator for cell death progression during pathogen response and senescence process in plants.

Role of Arabidopsis RabG3b and autophagy in tracheary element differentiation

The vascular system of plants consists of two conducting tissues, xylem and phloem, which differentiate from procambium cells. Xylem serves as a transporting system for water and signaling molecules and is formed by sequential developmental processes, including cell division/expansion, secondary cell wall deposition, vacuole collapse and programmed cell death (PCD). PCD during xylem differentiation is accomplished by degradation of cytoplasmic constituents, and it is required for the formation of hollow vessels, known as tracheary elements (TEs). Our recent study revealed that the small GTPase RabG3b acts as a regulator of TE differentiation through its autophagic activation. By using an Arabidopsis in vitro cell culture system, we showed that autophagy is activated during TE differentiation. Overexpression of a constitutively active RabG3b (RabG3bCA) significantly enhances both autophagy and TE differentiation, which are consistently suppressed in transgenic plants overexpressing a dominant negative form (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid-insensitive mutant bri1-301 and an autophagy mutant atg5-1. On the basis of our results, we propose that RabG3b functions as a component of autophagy and regulates TE differentiation by activating the process of PCD.

Overexpression of constitutively active Arabidopsis RabG3b promotes xylem development in transgenic poplars

An Arabidopsis small GTPase, RabG3b, was previously characterized as a component of autophagy and as a positive regulator for xylem development in Arabidopsis. In this work, we assessed whether RabG3b modulates xylem-associated traits in poplar in a similar way as in Arabidopsis. We generated transgenic poplars (Populus alba × Populus tremula var. glandulosa) overexpressing a constitutively active form of RabG3b (RabG3bCA) and performed a range of morphological, histochemical and molecular analyses to examine xylogenesis. RabG3bCA transgenic poplars showed increased stem growth due to enhanced xylem development. Autophagic structures were observed in differentiating xyelm cells undergoing programmed cell death (PCD) in wild-type poplar, and were more abundant in RabG3bCA transgenic poplar plants and cultured cells. Xylogenic activation was also accompanied by the expression of secondary wall-, PCD- and autophagy-related genes. Collectively, our results suggest that Arabidopsis RabG3b functions to regulate xylem growth through the activation of autophagy during wood formation in Populus, as does the same in Arabidopsis.

Evaluation of a transgenic poplar as a potential biomass crop for biofuel production

A transgenic poplar, in which the RabG3bCA gene from Arabidopsis was overexpressed, was analyzed for its biomass composition and enzymatic digestibility after chemical pretreatment. In comparison with a wild-type poplar (WT), the transgenic poplar (OX8) showed 9.8% higher glucan content. The levels of other biomass components did not differ greatly between WT and OX8. When WT and OX8 samples were pretreated by sulfuric acid (1%, w/v at 190 °C), sodium hydroxide (1%, w/v at 190 °C), or ammonia (14%, w/w at 80 °C), the washed pretreated solids of OX8 exhibited a higher enzymatic digestibility than those of WT in each chemical pretreatment. The sodium hydroxide pretreatment was the most effective among the three pretreatment processes, showing 58.7% and 69.4% of theoretical glucose yield from the saccharification of pretreated OX8 and WT, respectively. The transgenic poplar, growing faster and taller, was found to contain more glucan and have a higher enzymatic digestibility than WT.

Erratum to Role of an Arabidopsis Rab GTPase RabG3b in pathogen response and leaf senescence(Journal of Plant Biology, (2009), 52, 275 (275), 10.1007/s12374-009-9031-0)

In our previous proteomic analysis, we isolated a small GTPase RabG3b as a salicylic acid-responsive protein in Arabidopsis (Oh et al. in Plant Cell 17:2832–2847, 2005). Here, we constructed transgenic plants overexpressing wild-type (RabG3bOX), constitutively active (RabG3bCA), and dominant negative (RabG3bDN) forms of RabG3b for functional studies. The phenotypes of these transgenic plants were indistinguishable from wild-type plants under normal growth conditions. However, both RabG3bOX and RabG3bCA plants displayed unrestricted hypersensitive programmed cell death against a fungal toxin Fumonisin B1 and a fungal pathogen Alternaria brassicicola, whereas no major difference between wild-type and RabG3bDN plants was observed. In addition, RabG3bOX and RabG3bCA plants underwent accelerated leaf senescence compared to wild-type and RabG3bDN plants. These results suggest that RabG3b is a modulator for cell death progression during pathogen response and senescence process in plants.