Expansins: ever-expanding numbers and functions.

Expansins were first identified as cell-wall-loosening proteins that, at least in part, mediate pH-dependent extension of the plant cell wall and growth of the cell. More recently, it has been realized that expansins belong to two protein families, the alpha-and beta-expansins, and that they appear to be involved in regulating, besides cell expansion, a variety of plant processes, including morphogenesis, softening of fruits, and growth of the pollen tube of grasses through the stigma and the style. The Arabidopsis genome contains 26 alpha-expansin genes and the rice genome at least 26. There are more beta-expansin genes in monocots than in dicots, at least 14 in rice and five in Arabidopsis. Expansin genes are differentially regulated by environmental and hormonal signals, and hormonal regulatory elements have been found in their promoter regions. An analysis of exon/intron structure led to the hypothesis that alpha-and beta-expansins evolved from a common ancestral gene.

Expression of beta-expansins is correlated with internodal elongation in deepwater rice.

Fourteen putative rice (Oryza sativa) beta-expansin genes, Os-EXPB1 through Os-EXPB14, were identified in the expressed sequence tag and genomic databases. The DNA and deduced amino acid sequences are highly conserved in all 14 beta-expansins. They have a series of conserved C (cysteine) residues in the N-terminal half of the protein, an HFD (histidine-phenylalanine-aspartate) motif in the central region, and a series of W (tryptophan) residues near the carboxyl terminus. Five beta-expansin genes are expressed in deepwater rice internodes, with especially high transcript levels in the growing region. Expression of four beta-expansin genes in the internode was induced by treatment with gibberellin and by wounding. The wound response resulted from excising stem sections or from piercing pinholes into the stem of intact plants. The level of wound-induced beta-expansin transcripts declined rapidly 5 h after cutting of stem sections. We conclude that the expression of beta-expansin genes is correlated with rapid elongation of deepwater rice internodes, it is induced by gibberellin and wounding, and wound-induced beta-expansin mRNA appears to turn over rapidly.

Expression of two HOOKLESS genes in peas (Pisum sativum L.).

The apical hook of dark-grown dicotyledonous plants results from asymmetric growth of its inner and outer sides. It is a protective structure that prevents damage to the shoot apical meristem and the young leaves as the seedling pushes through the soil. Two phytohormones, ethylene and auxin, are thought to be involved in regulating apical hook formation. HOOKLESS1 (HLS1) of Arabidopsis was recognized as an ethylene-response gene whose product is required for hook formation. We cloned two cDNAs from peas, Ps-HLS1 and Ps-HLS2, whose products are functional homologs of HLS1. Both Ps-HLS1 and Ps-HLS2 complement the hls1 mutation in Arabidopsis. Expression of Ps-HLS1 is enhanced by ethylene and by IAA. Because the effect of ethylene is counteracted by 2,5-norbornadiene, an inhibitor of ethylene action, it appears that the primary factor in apical hook formation in peas is ethylene.

Isolation of a CONSTANS ortholog from Pharbitis nil and its role in flowering.

The short-day plant Pharbitis nil is a model plant for the study of photoperiodic control of floral initiation. Flower formation can be induced at the cotyledon stage by a single long night of at least 14 h in duration. Using differential display of mRNA we identified a P. nil ortholog of the Arabidopsis CONSTANS (CO) gene, which will be referred to as PnCO. Expression of PnCO was high after a 14-h night, but low when the dark period was 12 h or less. Our results indicate that the level of the PnCO transcript is photoperiodically regulated. After transfer from continuous light to darkness, PnCO showed a circadian pattern of expression. Expression of the CAB gene, which is a molecular marker for the circadian clock, exhibited a different pattern of expression than did PnCO and was not subject to the same photoperiodic control. A major portion of the PnCO transcripts contained an unspliced intron. Only the intron-free PnCO was able to complement the co mutant of Arabidopsis by shortening the time to flower.

Ethylene: a gaseous signal molecule in plants.

Ethylene regulates a multitude of plant processes, ranging from seed germination to organ senescence. Of particular economic importance is the role of ethylene as an inducer of fruit ripening. Ethylene is synthesized from S-adenosyl-L-methionine via 1-aminocyclopropane-1-carboxylic acid (ACC). The enzymes catalyzing the two reactions in this pathway are ACC synthase and ACC oxidase. Environmental and endogenous signals regulate ethylene biosynthesis primarily through differential expression of ACC synthase genes. Components of the ethylene signal transduction pathway have been identified by characterization of ethylene-response mutants in Arabidopsis thaliana. One class of mutations, exemplified by etr1, led to the identification of the ethylene receptors, which turned out to be related to bacterial two-component signaling systems. Mutations that eliminate ethylene binding to the receptor yield a dominant, ethylene-insensitive phenotype. CTR1 encodes a Raf-like Ser/Thr protein kinase that acts downstream from the ethylene receptor and may be part of a MAP kinase cascade. Mutants in CTR1 exhibit a constitutive ethylene-response phenotype. Both the ethylene receptors and CTR1 are negative regulators of ethylene responses. EIN2 and EIN3 are epistatic to CTR1, and mutations in either gene lead to ethylene insensitivity. Whereas the function of EIN2 in ethylene transduction is not known, EIN3 is a putative transcription factor involved in regulating expression of ethylene-responsive genes. Biotechnological modifications of ethylene synthesis and of sensitivity to ethylene are promising methods to prevent spoilage of agricultural products such as fruits, whose ripening is induced by ethylene.

A novel gibberellin-induced gene from rice and its potential regulatory role in stem growth.

Os-GRF1 (Oryza sativa-GROWTH-REGULATING FACTOR1) was identified in a search for genes that are differentially expressed in the intercalary meristem of deepwater rice (Oryza sativa L.) internodes in response to gibberellin (GA). Os-GRF1 displays general features of transcription factors, contains a functional nuclear localization signal, and has three regions with similarities to sequences in the database. One of these regions is similar to a protein interaction domain of SWI2/SNF2, which is a subunit of a chromatin-remodeling complex in yeast. The two other domains are novel and found only in plant proteins of unknown function. To study its role in plant growth, Os-GRF1 was expressed in Arabidopsis. Stem elongation of transformed plants was severely inhibited, and normal growth could not be recovered by the application of GA. Our results indicate that Os-GRF1 belongs to a novel class of plant proteins and may play a regulatory role in GA-induced stem elongation.

Alpha-expansins in the semiaquatic ferns Marsilea quadrifolia and Regnellidium diphyllum: evolutionary aspects and physiological role in rachis elongation.

To investigate the evolutionary history of expansins and their role in cell elongation in early land plants, we isolated two alpha-expansin genes, Mq-EXP1 and Rd-EXP1, respectively, from the semiaquatic ferns Marsilea quacdrifolia L. and Regnellidium diphyllum Lindm. The deduced amino acid sequences of the fern expansins exhibit a high degree of identity to those of seed plants, showing that expansin genes were conserved during the evolution of vascular plants. Gel-blot analysis of M. quadrifolia and R. diphyllum genomic DNA indicated that, in both ferns, alpha-expansins are encoded by multigene families. Expression of alpha-expansin genes probed with Mq-EXP1 was confined to the elongating region of the Marsilea rachis. Cell-wall proteins of M. quadrifolia induced in-vitro extension of acidified cucumber cell walls. In R. diphyllum, expression of Rd-EXP1 increased when elongation of the rachis was enhanced by submergence or ethylene. These results indicate that alpha-expansins act as wall-loosening proteins in ferns, as has been proposed for angiosperms. In addition, Rd-EXP1 may play a role in mediating elongation of the rachis in submerged plants.

Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase.

We identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active, autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

Differential regulation of genes encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase in etiolated pea seedlings: effects of indole-3-acetic acid, wounding, and ethylene.

Treatment of 5- to 6-day-old etiolated pea (Pisum sativum L.) seedlings with indole-3-acetic acid (IAA) induced within 15 min an increase in the transcript levels of two genes encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase, Ps-ACS1 and Ps-ACS2. Simultaneous treatment with ethylene inhibited this increase and also caused a decrease in ACC synthase enzyme activity as compared to that of seedlings treated with IAA alone. These results indicate that ethylene inhibits its own biosynthesis by decreasing ACC synthase transcript levels via a negative feedback loop. Wounding of pea stems had no effect on the expression of Ps-ACS1, but led within 10 min to an increase in the mRNA levels of Ps-ACS2. This increase was also inhibited by ethylene. The wound signal was transmitted over a distance of at least 4 cm through the stem with no delay in induction or response intensity. The rapid transmission of the wound response is consistent with the possibility that a hydraulic or electric signal is responsible for the spread of the wound response.

Tissue localization of expansins in deepwater rice.

Expansins are a family of proteins capable of inducing stress relaxation of isolated cell walls. In earlier studies, we showed that the expression of expansin genes in deepwater rice (Oryza sativa L.) is regulated by developmental, hormonal and environmental stimuli. Here, we describe the spatial distribution pattern of expansin transcripts and proteins in tissues and organs of deepwater rice using in situ mRNA hybridization and immunohistochemical analysis. Expansin transcripts and proteins are present at high levels in the growing internodal epidermis, which has thick cell walls and acts, therefore, as a growth-limiting cell layer. Expansins are also concentrated in the differentiating vascular bundles of internodes. In the primary root, expansins are predominantly expressed in the tip region, particularly in the epidermis, differentiating vascular cylinder, and around the pericyle. Developing adventitious roots and lateral root primordia also contain high levels of expansin mRNA. In the shoot apex, expansin transcripts are abundant in the emerging leaf primordia. Our results indicate that expansins play an important role in the expansion and differentiation of plant tissues and organs.

A gene encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase produces two transcripts: elucidation of a conserved response.

Indole-3-acetic acid (IAA) promotes ethylene biosynthesis in stems of etiolated pea (Pisum sativum L.) seedlings by rapidly increasing the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase mRNA and by enhancing the activity of the enzyme. Two cDNA clones encoding ACC synthase, Ps-ACS1 and Ps-ACS2, were isolated from a cDNA library prepared from the apical hooks of etiolated pea seedlings that had been treated with 100 microns IAA for 4 h. While studying the expression pattern of IAA-induced ACC synthase mRNA, we observed that the probe for Ps-ACS1 hybridized to two transcripts of 1.6 and 1.9 kb on RNA gel blots. The shorter transcript accumulated before the longer one did, indicating that it is not a degradation product of the latter. Because a similar observation, namely hybridization of one ACC synthase probe to two transcripts, has also been reported in other species, we investigated the relationship between the 1.6- and 1.9-kb transcripts. DNA gel blot analysis using the entire cDNA as probe and RNA gel blot analysis using the 3'-untranslated region as probe indicated that both transcripts are encoded by the same gene. Oligonucleotide-directed RNase H mapping showed that the transcripts differ in the sequence of their 5'-ends. Using 5'-RACE to obtain the DNA sequence of the shorter, transcript, we determined that the 1.6-kb transcript (Ps-ACS1b) begins within the second exon of the 1.9-kb transcript (Ps-ACS1a) and lacks the first 383 bases. Thus, Ps-ACS1b does not encode a full-length ACC synthase protein. Because the Ps-ACS1b sequence is identical to that of Ps-ACS1a, including proper splicing of the second intron, Ps-ACS1b appears to result from the use of an alternative, internal promoter.

Transcript level for a gene encoding a putative type 1a plasma membrane receptor is induced by gibberellin in deepwater rice.

In search for differentially expressed genes, a novel gene was identified whose transcript levels increased in response to gibberellin in the internodes of deepwater rice. Its expression was high in regions undergoing cell division and lower in the elongation and differentiation zones. Amino acid sequence analysis indicated that the gene may encode a type 1a receptor with an extracellular domain, a single transmembrane domain, and a short cytoplasmic domain.

Expression of expansin genes is correlated with growth in deepwater rice.

Expansins are a family of proteins that catalyze long-term extension of isolated cell walls. Previously, two expansin proteins have been isolated from internodes of deepwater rice, and three rice expansin genes, Os-EXP1, Os-EXP2, and Os-EXP3, have been identified. We report here on the identification of a fourth rice expansin gene, Os-EXP4, and on the expression pattern of the rice expansin gene family in deepwater rice. Rice expansin genes show organ-specific differential expression in the coleoptile, root, leaf, and internode. In these organs, there is increased expression of Os-EXP1, Os-EXP3, and Os-EXP4 in developmental regions where elongation occurs. This pattern of gene expression is also correlated with acid-induced in vitro cell wall extensibility. Submergence and treatment with gibberellin, both of which promote rapid internodal elongation, induced accumulation of Os-EXP4 mRNA before the rate of growth started to increase. Our results indicate that the expression of expansin genes in deepwater rice is differentially regulated by developmental, hormonal, and environmental signals and is correlated with cell elongation.

Expression of an ortholog of replication protein A1 (RPA1) is induced by gibberellin in deepwater rice.

Internodes of deepwater rice are induced to grow rapidly when plants become submerged. This adaptation enables deepwater rice to keep part of its foliage above the rising flood waters during the monsoon season and to avoid drowning. This growth response is, ultimately, elicited by the plant hormone gibberellin (GA). The primary target tissue for GA action is the intercalary meristem of the internode. Using differential display of mRNA, we have isolated a number of genes whose expression in the intercalary meristem is regulated by GA. The product of one of these genes was identified as an ortholog of replication protein A1 (RPA1). RPA is a heterotrimeric protein involved in DNA replication, recombination, and repair and also in regulation of transcription. A chimeric construct, in which the single-stranded DNA-binding domain of rice RPA1 was spliced into the corresponding region of yeast RPA1, was able to complement a yeast rpa1 mutant. The transcript level of rice RPA1 is high in tissues containing dividing cells. RPA1 mRNA levels increase rapidly in the intercalary meristem during submergence and treatment with GA before the increase in the level of histone H3 mRNA, a marker for DNA replication.

Expansins in deepwater rice internodes.

Cell walls of deepwater rice (Oryza sativa L.) internodes undergo long-term extension (creep) when placed under tension in acidic buffers. This is indicative of the action of the cell wall-loosening protein expansin. Wall extension had a pH optimum of around 4.0 and was abolished by boiling. Acid-induced extension of boiled cell walls could be reconstituted by addition of salt-extracted rice or cucumber cell wall proteins. Cucumber expansin antibody recognized a single protein band of 24.5-kD apparent molecular mass on immunoblots of rice cell wall proteins. Expansins were partially purified by concanavalin A affinity chromatography and sulfopropyl (SP) cation-exchange chromatography. The latter yielded two peaks with extension activity (SP20 and SP29), and immunoblot analysis showed that both of these active fractions contained expansin of 24.5-kD molecular mass. The N-terminal amino acid sequence of SP20 expansin is identical to that deduced from the rice expansin cDNA Os-EXP1. The N-terminal amino acid sequence of SP29 expansin matches that deduced from the rice expansin cDNA Os-EXP2 in six of eight amino acids. Our results show that two expansins occur in the cell walls of rice internodes and that they may mediate acid-induced wall extension.