Hyun Uk Kim

Ubiquitous and Endoplasmic Reticulum–Located Lysophosphatidyl Acyltransferase, LPAT2, Is Essential for Female but Not Male Gametophyte Development in Arabidopsis

Lysophosphatidyl acyltransferase (LPAT) is a pivotal enzyme controlling the metabolic flow of lysophosphatidic acid into different phosphatidic acids in diverse tissues. We examined putative LPAT genes in Arabidopsis thaliana and characterized two related genes that encode the cytoplasmic LPAT. LPAT2 is the lone gene that encodes the ubiquitous and endoplasmic reticulum (ER)–located LPAT. It could functionally complement a bacterial mutant with defective LPAT. LPAT2 and 3 synthesized in recombinant bacteria and yeast possessed in vitro enzyme activity higher on 18:1-CoA than on 16:0-CoA. LPAT2 was expressed ubiquitously in diverse tissues as revealed by RT-PCR, profiling with massively parallel signature sequencing, and promoter-driven β-glucuronidase gene expression. LPAT2 was colocalized with calreticulin in the ER by immunofluorescence microscopy and subcellular fractionation. LPAT3 was expressed predominately but more actively than LPAT2 in pollen. A null allele (lpat2) having a T-DNA inserted into LPAT2 was identified. The heterozygous mutant (LPAT2/lpat2) had minimal altered vegetative phenotype but produced shorter siliques that contained normal seeds and remnants of aborted ovules in a 1:1 ratio. Results from selfing and crossing it with the wild type revealed that lpat2 caused lethality in the female gametophyte but not the male gametophyte, which had the redundant LPAT3. LPAT2-cDNA driven by an LPAT2 promoter functionally complemented lpat2 in transformed heterozygous mutants to produce the lpat2/lpat2 genotype. LPAT3-cDNA driven by the LPAT2 promoter could rescue the lpat2 female gametophytes to allow fertilization to occur but not to full embryo maturation. Two other related genes, putative LPAT4 and 5, were expressed ubiquitously albeit at low levels in diverse organs. When they were expressed in bacteria or yeast, the microbial extract did not contain LPAT activity higher than the endogenous LPAT activity. Whether LPAT4 and 5 encode LPATs remains to be elucidated.

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.

Plastid Lysophosphatidyl Acyltransferase Is Essential for Embryo Development in Arabidopsis

Lysophosphatidyl acyltransferase (LPAAT) is a pivotal enzyme controlling the metabolic flow of lysophosphatidic acid into different phosphatidic acids in diverse tissues. A search of the Arabidopsis genome database revealed five genes that could encode LPAAT-like proteins. We identified one of them, LPAAT1, to be the lone gene that encodes the plastid LPAAT. LPAAT1 could functionally complement a bacterial mutant that has defective LPAAT. Bacteria transformed with LPAAT1 produced LPAAT that had in vitro enzyme activity much higher on 16:0-coenzyme A than on 18:1-coenzyme A in the presence of 18:1-lysophosphatidic acid. LPAAT1 transcript was present in diverse organs, with the highest level in green leaves. A mutant having a T-DNA inserted into LPAAT1 was identified. The heterozygous mutant has no overt phenotype, and its leaf acyl composition is similar to that of the wild type. Selfing of a heterozygous mutant produced normal-sized and shrunken seeds in the Mendelian ratio of 3:1, and the shrunken seeds could not germinate. The shrunken seeds apparently were homozygous of the T-DNA-inserted LPAAT1, and development of the embryo within them was arrested at the heart-torpedo stage. This embryo lethality could be rescued by transformation of the heterozygous mutant with a 35S:LPAAT1 construct. The current findings of embryo death in the homozygous knockout mutant of the plastid LPAAT contrasts with earlier findings of a normal phenotype in the homozygous mutant deficient of the plastid glycerol-3-phosphate acyltransferase; both mutations block the synthesis of plastid phosphatidic acid. Reasons for the discrepancy between the contrasting phenotypes of the two mutants are discussed.

Genome-wide identification of glucosinolate synthesis genes in Brassica rapa.

Glucosinolates play important roles in plant defense against herbivores and microbes, as well as in human nutrition. Some glucosinolate‐derived isothiocyanate and nitrile compounds have been clinically proven for their anticarcinogenic activity. To better understand glucosinolate biosynthesis in Brassica rapa, we conducted a comparative genomics study with Arabidopsis thaliana and identified total 56 putative biosynthetic and regulator genes. This established a high colinearity in the glucosinolate biosynthesis pathway between Arabidopsis and B. rapa. Glucosinolate genes in B. rapa share 72–94% nucleotide sequence identity with the Arabidopsis orthologs and exist in different copy numbers. The exon/intron split pattern of B. rapa is almost identical to that of Arabidopsis, although inversion, insertion, deletion and intron size variations commonly occur. Four genes appear to be nonfunctional as a result of the presence of a frame shift mutation and retrotransposon insertion. At least 12 paralogs of desulfoglucosinolate sulfotransferase were found in B. rapa, whereas only three were found in Arabidopsis. The expression of those paralogs was not tissue‐specific but varied greatly depending on B. rapa tissue types. Expression was also developmentally regulated in some paralogs but not in other paralogs. Most of the regulator genes are present as triple copies. Accordingly, glucosinolate synthesis and regulation in B. rapa appears to be more complex than that of Arabidopsis. With the isolation and further characterization of the endogenous genes, health‐beneficial vegetables or desirable animal feed crops could be developed by metabolically engineering the glucosinolate pathway.

New pollen-specific receptor kinases identified in tomato, maize and Arabidopsis: the tomato kinases show overlapping but distinct localization patterns on pollen tubes.

We previously characterized LePRK1 and LePRK2, pollen-specific receptor kinases from tomato (Muschietti et al., 1998). Here we identify a similar receptor kinase from maize, ZmPRK1, that is also specifically expressed late in pollen development, and a third pollen receptor kinase from tomato, LePRK3. LePRK3 is less similar to LePRK1 and LePRK2 than either is to each other. We used immunolocalization to show that all three LePRKs localize to the pollen tube wall, in partially overlapping but distinct patterns. We used RT-PCR and degenerate primers to clone homologues of the tomato kinases from other Solanaceae. We deduced features diagnostic of pollen receptor kinases and used these criteria to identify family members in the Arabidopsis database. RT-PCR confirmed pollen expression for five of these Arabidopsis candidates; two of these are clearly homologues of LePRK3. Our results reveal the existence of a distinct pollen-specific receptor kinase gene family whose members are likely to be involved in perceiving extracellular cues during pollen tube growth.

Endoplasmic Reticulum-Located PDAT1-2 from Castor Bean Enhances Hydroxy Fatty Acid Accumulation in Transgenic Plants

Ricinoleic acid (12-hydroxy-octadeca-9-enoic acid) is a major unusual fatty acid in castor oil. This hydroxy fatty acid is useful in industrial materials. This unusual fatty acid accumulates in triacylglycerol (TAG) in the seeds of the castor bean (Ricinus communis L.), even though it is synthesized in phospholipids, which indicates that the castor plant has an editing enzyme, which functions as a phospholipid:diacylglycerol acyltransferase (PDAT) that is specific to ricinoleic acid. Transgenic plants containing fatty acid Δ12-hydroxylase encoded by the castor bean FAH12 gene produce a limited amount of hydroxy fatty acid, a maximum of around 17% of TAGs present in Arabidopsis seeds, and this unusual fatty acid remains in phospholipids of cell membranes in seeds. Identification of ricinoleate-specific PDAT from castor bean and manipulation of the phospholipid editing system in transgenic plants will enhance accumulation of the hydroxy fatty acid in transgenic seeds. The castor plant has three PDAT genes; PDAT1-1 and PDAT2 are homologs of PDAT, which are commonly found in plants; however, PDAT1-2 is newly grouped as a castor bean-specific gene. PDAT1-2 is expressed in developing seeds and localized in the endoplasmic reticulum, similar to FAH12, indicating its involvement in conversion of ricinoleic acid into TAG. PDAT1-2 significantly enhances accumulation of total hydroxy fatty acid up to 25%, with a significant increase in castor-like oil, 2-OH TAG, in seeds of transgenic Arabidopsis, which is an identification of the key gene for oilseed engineering in production of unusual fatty acids.

The AAE14 gene encodes the Arabidopsis o‐succinylbenzoyl‐CoA ligase that is essential for phylloquinone synthesis and photosystem‐I function

Phylloquinone is the one-electron carrier at the A(1) site of photosystem I, and is essential for photosynthesis. Arabidopsis mutants deficient in early steps of phylloquinone synthesis do not become autotrophic and are seedling lethals, even when grown on sucrose-supplemented media. Here, we identify acyl-activating enzyme 14 (AAE14, At1g30520) as the o-succinylbenzoyl-coenzyme A (OSB-CoA) ligase acting in phylloquinone synthesis. Three aae14 mutant alleles, identified by reverse genetics, were found to be seedling lethal, to contain no detectable phylloquinone (< 0.1 pmol mg(-1) fresh weight) compared with 10 pmol mg(-1) fresh weight in wild-type leaves, and to accumulate OSB. AAE14 was able to restore menaquinone biosynthesis when expressed in an Escherichia coli mutant disrupted in the menE gene that encodes the bacterial OSB-CoA ligase. Weak expression of an AAE14 transgene in mutant plants (controlled by the uninduced XVE promoter) resulted in chlorotic, slow-growing plants that accumulated an average of 4.7 pmol mg(-1) fresh weight of phylloquinone. Inducing the XVE promoter in these plants, or expressing an AAE14 transgene under the control of the CaMV 35S promoter, led to full complementation of the mutant phenotype. aae14-mutant plants were also able to synthesize phylloquinone when provided with 1,4-dihydroxy-2-naphthoate, an intermediate in phylloquinone synthesis downstream of the OSB-CoA ligase reaction. Expression of an AAE14:GFP reporter construct indicated that the protein accumulated in discrete foci within the chloroplasts. This and other evidence suggests that the enzymes of phylloquinone synthesis from isochorismate may form a complex in the chloroplast stroma to facilitate the efficient channeling of intermediates through the pathway.

Constitutive and seed-specific expression of a maize lysine-feedback-insensitive dihydrodipicolinate synthase gene leads to increased free lysine levels in rice seeds

In order to improve the nutritional value of rice, we prepared transgenic rice plants with a lysine-feedback-insensitive maize dhps gene under the control of CaMV 35S and the rice glutelin GluB-1 promoter for over-expression and seed-specific expression. The transgenic plants were fertile and expressed the dhps gene abundantly or specifically in rice seeds. The transgenic lines (TC lines) containing mutated dhps controlled by CaMV 35S promoter possessed higher mutated DHPS transcript levels and in vitro DHPS activities in seeds than those of TS lines containing the mutated dhps gene driven by a seed-specific promoter, GluB-1. The content of free lysine in immature seeds of both TC and TS lines was higher than that of wild-type plants. The content of free lysine in mature seeds of TC lines was still higher than, but that of TS lines was similar to, that of wild-type plants. From a comparison of DHPS and lysine-ketoglutarate reductase (LKR) expression levels we conclude that the presence of the foreign dhps gene leads to an increase of LKR activity, resulting in enhanced lysine catabolism. However, over-expression of the mutant dhps gene in a constitutive manner overcomes lysine catabolism and sustains a high lysine level in mature rice seeds.

Induction of male sterile cabbage using a tapetum-specific promoter from Brassica campestris L. ssp. pekinensis

The anther (tapetum)-specific gene BcA9 was isolated from Chinese cabbage, Brassica campestris L. ssp. pekinensis cv. Jangwon, using the Arabidopsis tapetum-specific A9 gene as a probe. The DNA and amino acid sequences of the coding region of the BcA9 gene showed high homology with A9 genes from Arabidopsis and B. napus. However, the DNA sequences of the 5′ noncoding (promoter) region were different, except for the sequence from −281 to −89. To test the specific activity of this promoter, a plant expression vector, pGR011, was constructed by fusing the BcA9 promoter and the cytotoxic diphtheria toxin A-chain (DTx-A) gene. Several transgenic plants from cabbage, B. oleracea ssp. capitata, were obtained by way of Agrobacterium-mediated transformation. Southern blot analysis indicated that the tapetum-specific BcA9 promoter and DTx-A gene were successfully integrated into the genome of the transgenic cabbage. Under the control of the BcA9 promoter, expression of the cytotoxic DTx-A gene in the tapetal cells of the transgenic plants resulted in male sterile cabbages. Microscopic examination revealed that pollen grains in anthers of the male sterile cabbages had not developed normally, but the vegetative growth and phenotype showed no difference compared to wild-type plants.

Malonyl-CoA Synthetase, Encoded by ACYL ACTIVATING ENZYME13, Is Essential for Growth and Development of Arabidopsis

Malonic acid inhibits succinate dehydrogenase and is toxic to Arabidopsis seedlings. Biochemical assays demonstrate that ACYL ACTIVATING ENZYME13 (AAE13) of Arabidopsis is a malonyl-CoA synthetase. Characterization of an aae13 mutant indicates that the malonyl-CoA synthetase encoded by AAE13 is required for detoxification of endogenous malonate and for healthy growth and development of plants. Malonyl-CoA is the precursor for fatty acid synthesis and elongation. It is also one of the building blocks for the biosynthesis of some phytoalexins, flavonoids, and many malonylated compounds. In plants as well as in animals, malonyl-CoA is almost exclusively derived from acetyl-CoA by acetyl-CoA carboxylase (EC 6.4.1.2). However, previous studies have suggested that malonyl-CoA may also be made directly from malonic acid by malonyl-CoA synthetase (EC 6.2.1.14). Here, we report the cloning of a eukaryotic malonyl-CoA synthetase gene, Acyl Activating Enzyme13 (AAE13; At3g16170), from Arabidopsis thaliana. Recombinant AAE13 protein showed high activity against malonic acid (Km = 529.4 ± 98.5 μM; Vm = 24.0 ± 2.7 μmol/mg/min) but little or no activity against other dicarboxylic or fatty acids tested. Exogenous malonic acid was toxic to Arabidopsis seedlings and caused accumulation of malonic and succinic acids in the seedlings. aae13 null mutants also grew poorly and accumulated malonic and succinic acids. These defects were complemented by an AAE13 transgene or by a bacterial malonyl-CoA synthetase gene under control of the AAE13 promoter. Our results demonstrate that the malonyl-CoA synthetase encoded by AAE13 is essential for healthy growth and development, probably because it is required for the detoxification of malonate.