The idea that a specific chemical stimulus is involved in some processes of plant development was introduced by European plant scientists in the early 20th century. After the discovery of auxin by Went in 1926, this hormone became a subject of intensive studies in the physiology of plant growth and development. In Japan, studies on auxin started in the middle of the 1930s. At that time, gibberellin was discovered in Japan. Unfortunately, gibberellin was regarded as a fungal toxin and no special attention was paid to its physiological significance. The first paper on auxin in Japan was published by Masayuki Nagao in 1936, dealing with its transport in the root, which was then a subject of dispute. Before World War II (~1940) there were only about ten auxin researchers, excluding those in applied areas. In this article I will introduce their studies and some of the applied studies. I will also pay special attention to a series of work by Sadao Yasuda who was a pioneer researcher of plant reproductive physiology in Japan. He studied self-incompatibility (1927–1932) and parthenocarpy (1930–1940) which are related to plant growth substances. He demonstrated that a special substance (inhibitor) was involved in self-incompatibility. This is probably the first case in Japan for demonstrating the involvement of a regulatory inhibitor in plant development. In his study on parthenocarpy he concluded that “pollen hormone” and auxin are identical in their physiological nature.
Plants are constantly exposed to environmental factors, including biotic and abiotic stresses, which may confer serious damage and affect survival. In order to cope with these stresses, plants have evolved a variety of defense systems. Despite intensive surveys, the molecular mechanisms are still not completely understood, and in particular, information on diversification of proteins is limited. In this article, we focus on examples of proteins changing both cellular localization and functions. Experimentally, six such proteins have so far been identified, all of them containing motifs of nuclear transcription factors, but localized in plastids: a 41-kDa protein from Nicotiana tabacum containing a zinc finger motif, shown to be a chloroplast nucleoid DNA binding protein (CND41); a plastid envelope DNA binding protein (PEND) from Pisum sativum possessing a basic domain plus leucine zipper motif; proteins designated as PD1 and PD3 also isolated from P. sativum, both having AT-hook motifs; a protein with a basic helix-loop-helix (bHLH) motif from Arabidopsis thaliana proposed to be a plastid transcription factor, and designated as PTF1; one wound-induced protein from N. tabacum (NtWIN4) shown to be a bHLH protein and exclusively localized into plastids. Judging from their structures, these proteins could have originated from eukaryotic ancestors. At the N-terminus, they possess clustered basic residues, which might constitute the key structure for conversion from nuclear transcription factors to plastid-resident proteins.
The unique mutant nolac-K4 (non-organogenic callus with loosely attached cells), which was generated by T-DNA transformation using leaf-disk cultures of haploid Nicotiana plumbaginifolia, has lost the ability to form adventitious shoots and also shows decreased intercellular attachment. The gene tagged with the T-DNA in this line, named NpLRX1 (LRR-EXTENSIN 1), is a novel tobacco gene that encodes a cell-wall protein containing chimeric leucine-rich repeat and extensin domains. The gene is highly similar to the Arabidopsis LRX genes, and phylogenetic analysis places it in the vegetative LRX clade. NpLRX1 is expressed ubiquitously in vegetative tissues, strongly in the leaf and root vascular bundles, and in emerging lateral roots and root tips. Tobacco leaf disks transformed with an NpLRX1-RNAi construct displayed aberrant adventitious buds and disorganized cell morphogenesis with large intercellular spaces. The shapes of NpLRX1-RNAi-transformed BY-2 cells were irregular, and the cells showed disorganized cortical microtubules. These results suggest that NpLRX1, the lack of which may be responsible for the nolac-K4 mutation, is a new tobacco LRX gene that has regulatory roles in cell morphogenesis that are essential for plant tissue development.
UV-irradiated cotyledonary protoplasts of Solanum integrifolium and iodoacetamide-treated cotyledonary protoplasts of S. sanitwongsei were electrofused and cultured. Regenerated plants were classified into three groups based on morphology and genomic in situ hybridization data. Morphology of the first group was intermediate between those of parental species. The plants bore fruits with viable seeds and had a chromosome number of 2n=48, the sum of the parental chromosome numbers, suggesting that they were symmetric fusion hybrids. Morphology of the plants in the second group was more S. integrifolium-like than that of the first group, and had two sets of S. integrifolium chromosomes and one set of S. sanitwongsei chromosomes. In contrast, plants in the third group had one set of S. integrifolium chromosomes and two sets of S. sanitwongsei chromosomes. Plants in the second and third groups were less vigorous than those in the first group, and bore few fruits. Electrophoretic analysis of the isozymes shikimate dehydrogenase, isocitrate dehydrogenase, and phosphoglucomutase, as well as random amplified polymorphic DNA analysis, demonstrated that 23 of regenerated plants from the three groups were somatic hybrids. The plants in the first group grew more vigorously than the parental plants and produced more than 5000 seeds per plant. The fertile somatic hybrids obtained in this study may be suitable candidates for eggplant rootstocks.
Many flowering plants possess self-incompatibility (SI) systems to prevent inbreeding. In Brassica, SI recognition is controlled by the multiallelic gene complex (S-haplotypes) at the S-locus, which encodes both the male determinant (S-locus protein 11 (SP11/SCR)) and the female determinant (S-receptor kinase (SRK)). After self-pollination, an S-haplotype specific interaction between pollen-borne SP11 and its cognate stigmatic SRK receptor induces SI signaling in stigma papilla cells, which results in the rejection of the self-pollen. Previous genetic analysis by our group of a self-compatible mutant revealed the involvement of a membrane-anchored cytoplasmic protein kinase, M-locus protein kinase (MLPK), in SI signaling. The plasma membrane localization of MLPK suggests that it functions in the vicinity of SRK, but the physiological relationship between the two proteins remains unknown. In the present study, we looked for a direct interaction between SRK and MLPK using both genetic and biochemical techniques. Although a conventional yeast two-hybrid system and a split-ubiquitin membrane-based yeast two-hybrid system failed to detect a direct interaction between SRK and MLPK, an in vitro phosphorylation assay indicated that the kinase domain of SRK could efficiently phosphorylate MLPK. These results suggest that MLPK could be a direct target of SRK in Brassica SI signaling.
The hypersensitive response (HR) is a form of programmed cell death commonly associated with immune response in plants. Incompatible N1141 strain of Acidovorax avenae elicits HR cell death in cultured rice cells, while flagellin-deficient N1141 mutant (Δfla-N1141) lost the ability to induce immune responses. We present evidence that initiation of HR cell death induced by the incompatible N1141 strain requires de novo protein synthesis. The protein kinase inhibitors, staurosporine and K252a, effectively suppressed HR cell death, while the protein phosphatase inhibitors, calyculin A and okadaic acid, did not affect the induction of HR cell death. To identify the key initiator of HR cell death, PCR subtraction analysis and microarray analysis were performed. PCR subtraction analysis identified 42 genes which are induced during HR cell death, and microarray analysis (22K) showed that 87 genes were induced during HR cell death. Among identified genes, OsNAC gene encoding plant specific transcription factor was identified in both analyses. Real-time reverse-transcription PCR analysis for several OsNAC family genes revealed that OsNAC4 gene was strongly and specifically induced during HR cell death in cultured rice cells. Results showed that OsNAC4 transcription factor is an influential candidate as a key initiator in plant HR cell death.
The short-day plant Pharbitis [Ipomoea] nil has been a model plant in physiological studies of photoperiodic control of floral initiation. However, molecular mechanisms underlying photoperiodic responses, including the induction of flowering in P. nil, are largely unknown. Here we identified and characterized cDNAs whose expression patterns were different in short- and long-day conditions, based on fluorescent differential display and RNA gel blot analysis, to gain insight into the molecular mechanisms of photoperiodic responses in P. nil. The cDNA clones included genes encoding ADP-glucose pyrophosphorylase, a putative protein kinase, eukaryotic initiation factor eIF-4, a putative aldehyde dehydrogenase, subtilisin-like proteinase, crooked neck (crn)-like protein, and cryptochrome 1. The possible roles of these genes in the photoperiodic response, including the control of flowering time in P. nil, are discussed.
An ATPase associated with various cellular activities (AAA) protein was previously shown to be involved in pathogen response in tobacco plants and designated as NtAAA1. Transgenic tobacco lines in which NtAAA1 was suppressed by the RNA-interference (RNAi) were found to exhibit an elevated resistance to Pseudomonas syringae infection, suggesting that NtAAA1 negatively controlled the defense reaction. To identify genes that were regulated by NtAAA1, differential micro-array screening between NtAAA1-RNAi and wild type plants was performed. Results brought out 330 affected genes, which were classified into functional categories, including transcriptional regulation, signal transduction, secondary metabolism and others. Notably, 43 genes were stress- and defense-related, among which 10 were phytohormone-related. Subsequent examination revealed that, in RNAi transgenic plants, genes related to salicylic acid were up-regulated, whereas those related to jasmonic acid and ethylene were generally down-regulated. When salicylic acid was exogenously applied to leaves, expression of PR-1a, a maker gene of pathogen response, was evidently induced at much higher level in NtAAA1-RNAi transgenic lines than in the control. Simultaneous application of jasmonic acid with salicylic acid markedly cancelled the effect of salicylic acid in the control, but not much in NtAAA1-RNAi transgenic plants. The present findings suggested that NtAAA1 broadly functions in cellular metabolism, and particularly that, responding to jasmonic acid and/or ethylene signals, it might interfere with salicylic acid signaling. This system maintains the defense response at appropriate levels, so that detrimental necrosis is avoided, and therefore NtAAA1 may be regarded as a molecular switch of the salicylic acid signaling pathway.
We previously showed the high expression of a 23 kD protein (P23k) in germinating barley seeds. In this study, we examined whether P23k is involved in supplying sugar to developing barley grains. RT-PCR analysis revealed that, although P23k expression was not detectable in developing grain, the 23 kD jasmonate-induced protein (JIP-23) exhibited a high level of RNA expression. JIP-23 belongs to the same protein family as P23k. Its remarkable expression was specifically confirmed at early development stage when sugar import into the filling grains is active. Furthermore, in situ hybridization and immunohistochemistry showed that JIP-23 is localized to active tissues in sugar import, such as vascular tissues, nucellar projections, endosperm transfer cells, and inter nucellar cells. Taken together, these results suggest that JIP-23 is related to sugar import into filling grain of developing barley seeds. Hence, we propose that jasmonate may regulate the development of barley seed.
The micropropagation protocols for Aralia elata and Phellodendron amurense were established. The survival rates in the acclimatization process between the two species were found to be greatly different. To find the reasons for this difference, the morphological and histological changes in the leaf during successive micropropagation stages in in vitro-derived plantlets of A. elata and P. amurense were compared. Significant differences in mesophyll development in differentiation as well as vascular system development were found during the root induction stages. Such different leaf structures may be responsible for the survival rate through the acclimatization process. These results suggested that changes in leaf structure from in vitro to ex vitro plants are important for histological development, rendering them appropriate for in vitro hardening, which begins at the root elongation stage.
Low-glutelin rice has low content of digestible protein glutelin, and is expected to use as raw material for sake brewing. To characterize endosperm cell structure of low-glutelin rice, the storage proteins of endosperm cells in polished rice (degree of milling, 70%; used as raw material for sake brewing) of two low-glutelin rice varieties, LGC1 and Tashu-kei 1001, and two general rice cultivars (brewer's rice), Hyogokitanishiki and Yamadanishiki, were observed using a transmission electron microscope. Low-glutelin rice differed from general rice cultivars in the composition of major storage proteins and the transgranular distribution of storage proteins as well as in the distribution of protein bodies in endosperm cells. Furthermore, low-glutelin rice had a specific endosperm cell structure containing many type I protein bodies even in the outer layer of polished rice grains.
Storage proteins in cotyledons of legume plants are degraded by proteinases after germination. We examined whether abscisic acid (ABA), gibberellin (GA) and brassinosteroid (BR) are involved in the expression of cysteine proteinases in cotyledons of common bean seeds with RNA blotting using cDNAs for five papain-like proteinases, EP-C1, CP1, CP2, CP3, CP4 and two legumain-like proteinases, LLP1 and LLP2 as probes. Six genes, except for CP4, are activated after seed imbibition. These germination-induced cysteine proteinase genes (GICPs) are repressed by exogenously applied (+)-5′α, 8′-cyclo-ABA which is more resistant to hydroxylation than ABA. Either GA3 or epi-BR activated GICPs in the presence of ABA, suggesting that both of GA and BR are involved in the expression of GICPs. It is possible that GA biosynthesis is required for the activation of GICPs, because either GA-biosynthesis inhibitors, chloroethyltrimethyl-ammonium chloride or prohexadione calcium repressed GICPs and this repression was restored by exogenously applied GA3.
EARLY FLOWERING 3 (ELF3) plays key roles in the control of circadian rhythms, photoperiodic flowering, hypocotyls length and response to light in Arabidopsis. ELF3, however, encodes a protein without any motifs or domains of known function. Biochemical function of ELF3 has been largely unknown. Here we show the first demonstration of diversity of numbers of polyglutamines in the ELF3 among 60 Arabidopsis wild-type accessions. Significant correlation of the numbers of polyglutamines in ELF3 with two parameters of circadian rhythms, period and phase, suggest that the length of polyglutamine tract may affect the functions of ELF3 in the control of circadian rhythms.
Five cDNA clones encoding 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) were isolated from tulip (Tulipa gesneriana L.) by differential screening of the petal cDNA library and were designated as TgACO1, TgACO2, TgACO3, TgACO4 and TgACO5. The deduced amino acid sequences exhibited similarity to ACO proteins from other plant species. Among these proteins, TgACO1-4 have high similarity (90–94% identity) each other, whereas TgACO5 showed low similarity compared with the other four proteins (50% identity). Phylogenic analysis indicated that TgACO1-4 and TgACO5 are distant. Genomic analysis of TgACO1-4 showed that they are organized into four exons and three introns, whereas TgACO5 consists of three exons interrupted by two introns. Real-time RT-PCR analysis of gene expression revealed that TgACO1, TgACO3 and TgACO5 were expressed in wilting petals, leaves, and stems, respectively, whereas TgACO2 and TgACO4 were expressed only at basal levels in these tissues. Therefore, tulip ACO genes seem to be regulated differentially among the vegetative tissues and during flower senescence.
An antimicrobial protein was purified from leaves of Wasabia japonica L., and designated as WjAMP-2. WjAMP-2 strongly inhibited growth of four phytopathogenic fungi. The deduced amino acid sequence from WjAMP-2 cDNA showed a homology with pathogenesis-related protein 1 (PR-1) from several higher plants. Expression of WjAMP-2 was induced by inoculation with fungal pathogens and with treatments by salicylic acid, but not by methyl jasmonate. Overexpression of WjAMP-2 in tobacco plants conferred resistance against Botrytis cinerea. These results suggest that WjAMP-2 (PR-1) is an antifungal protein with a defensive role against phytopathogenic fungi in wasabi plants.
The oligosaccharide structures of total glycoproteins in suspension-cultured Arabidopsis thaliana MM2d cells are determined. The N-linked sugar chains released by hydrazinolysis were labeled with 2-aminopyridine, and analyzed by a combination of HPLC, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and exoglucosidase digestion. The glycan structures determined were GlcNAcMan3FucXylGlcNAc2 (8.5%), Man3FucXylGlcNAc2 (70.0%), Man3XylGlcNAc2 (13.7%), and GlcNAcMan3XylGlcNAc2 (7.8%). All the glycan structures of glycoproteins from Arabidopsis MM2d cells used in this study contain typical plant bisecting either β1,2-xylose or α1,3-fucose residues, showing that suspension-cultured MM2d cells have a glycosylation potential similar to that of tobacco BY2 suspension-cultured cells.
Plant-based assays for monitoring contaminated environments provide inexpensive and nontechnical means of environmental analysis. Here we report a model system for monitoring selenium and chromium, which are highly toxic heavy metals for living organisms. The major forms of selenium and chromium in nature are selenate and chromate. As toxic analogs of sulfate, they cause sulfur deficiency in plants by inhibiting the uptake of sulfate from the environment. We used a fusion gene construct consisting of a sulfur-responsive promoter region of the high-affinity sulfate transporter SULTR1;2 from Arabidopsis and green fluorescent protein (GFP; PSULTR1;2-GFP) to quantify the levels of selenate and chromate by GFP accumulation. The PSULTR1;2-GFP transgenic Arabidopsis plants showed drastic increases in GFP with the addition of selenate or chromate to the medium. The increase in GFP was concentration-dependent relative to the amounts of contaminants in the medium, suggesting the potential of PSULTR1;2-GFP plants as indicators in quantifying environmental selenate and chromate.