Regulation of seed germination and seedling growth by an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytocystatin isoform, <Emphasis Type="Italic">AtCYS6</Emphasis>

Phytocystatins are cysteine proteinase inhibitors in plants that are implicated in the endogenous regulation of protein turnover and defense mechanisms against insects and pathogens. A cDNA encoding a phytocystatin called AtCYS6 (Arabidopsis thaliana phytocystatin6) has been isolated. We show that AtCYS6 is highly expressed in dry seeds and seedlings and that it also accumulates in flowers. The persistence of AtCYS6 protein expression in seedlings was promoted by abscisic acid (ABA), a seed germination and post-germination inhibitory phytohormone. This finding was made in transgenic plants bearing an AtCYS6 promoter–β-glucuronidase (GUS) reporter construct, where we found that expression from the AtCYS6 promoter persisted after ABA treatment but was reduced under control conditions and by gibberellin₄₊₇ (GA₄₊₇) treatment during the germination and post-germinative periods. In addition, constitutive over-expression of AtCYS6 retarded germination and seedling growth, whereas these were enhanced in an AtCYS6 knock-out mutant (cys6-2). Additionally, cysteine proteinase activities stored in seeds were inhibited by AtCYS6 in transgenic Arabidopsis. From these data, we propose that AtCYS6 expression is enhanced by the germination inhibitory phytohormone ABA and that it participates in the control of germination rate and seedling growth by inhibiting the activity of stored cysteine proteinases.     

Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases

Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais.     

Heterologous expression of taro cystatin protects transgenic tomato against Meloidogyne incognita infection by means of interfering sex determination and suppressing gall formation

Plant-parasitic nematodes are a major pest of many plant species and cause global economic loss. A phytocystatin gene, Colocasia esculenta cysteine proteinase inhibitor (CeCPI), isolated from a local taro Kaosiang No. 1, and driven by a CaMV35S promoter was delivered into CLN2468D, a heat-tolerant cultivar of tomato (Solanum lycopersicum). When infected with Meloidogyne incognita, one of root-knot nematode (RKN) species, transgenic T₁ lines overexpressing CeCPI suppressed gall formation as evidenced by a pronounced reduction in gall numbers. In comparison with wild-type plants, a much lower proportion of female nematodes without growth retardation was observed in transgenic plants. A decrease of RKN egg mass in transgenic plants indicated seriously impaired fecundity. Overexpression of CeCPI in transgenic tomato has inhibitory functions not only in the early RKN infection stage but also in the production of offspring, which may result from intervention in sex determination.     

Suppression of telomere-binding protein gene expression represses seed and fruit development in tomato

Tomato (Solanum lycopersicum L.) plants were transformed with an antisense construct of a cDNA encoding tomato telomere-binding protein (LeTBP1) to describe the role of a telomere-binding protein at the whole plant level. Fruit size decreased corresponding to the degree of suppression of LeTBP1 expression. This inhibition of fruit development was likely due to a decrease in the number of seeds in the LeTBP1 antisense plants. Pollen fertility and pollen germination rate decreased in accordance with the degree of suppression of LeTBP1 expression. Ovule viability was also reduced in the LeTBP1 antisense plants. Although plant height was somewhat reduced in the antisense plants compared to the control plants, the number and weight of leaves were unaffected by LeTBP1 suppression. The number and morphology of flowers were also normal in the antisense plants. These indicate that reduced fertility in the antisense plants is not an indirect effect of altered vegetative growth. LeTBP1 expression was sensitive to temperature stress in wild-type plants. We conclude that LeTBP1 plays a critical role in seed and fruit development rather than vegetative growth and flower formation.     

Three zinc‐finger RNA‐binding proteins in cabbage (Brassica rapa) play diverse roles in seed germination and plant growth under normal and abiotic stress conditions

Despite the increasing understanding of the stress‐responsive roles of zinc‐finger RNA‐binding proteins (RZs) in several plant species, such as Arabidopsis thaliana, wheat (Triticum aestivum) and rice (Oryza sativa), the functions of RZs in cabbage (Brassica rapa) have not yet been elucidated. In this study, the functional roles of the three RZ family members present in the cabbage genome, designated as BrRZ1, BrRZ2 and BrRZ3, were investigated in transgenic Arabidopsis under normal and environmental stress conditions. Subcellular localization analysis revealed that all BrRZ proteins were exclusively localized in the nucleus. The expression levels of each BrRZ were markedly increased by cold, drought or salt stress and by abscisic acid (ABA) treatment. Expression of BrRZ3 in Arabidopsis retarded seed germination and stem growth and reduced seed yield of Arabidopsis plants under normal growth conditions. Germination of BrRZ2‐ or BrRZ3‐expressing Arabidopsis seeds was delayed compared with that of wild‐type seeds under dehydration or salt stress conditions and cold stress conditions, respectively. Seedling growth of BrRZ3‐expressing transgenic Arabidopsis plants was significantly inhibited under salt, dehydration or cold stress conditions. Notably, seedling growth of all three BrRZ‐expressing transgenic Arabidopsis plants was inhibited upon ABA treatment. Importantly, all BrRZs possessed RNA chaperone activity. Taken together, these results indicate that the three cabbage BrRZs harboring RNA chaperone activity play diverse roles in seed germination and seedling growth of plants under abiotic stress conditions as well as in the presence of ABA.     

Molecular cloning,recombinant gene expression,and antifungal activity of cystatin from taro (<Emphasis Type="Italic">Colocasia esculenta</Emphasis> cv. Kaosiung no. 1)

A cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from taro corms (Colocasia esculenta) using both degenerated primers/RT-PCR amplification and 5-/3-RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 206 amino acid residues, with a deduced molecular weight of 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to Eudicots rather than to Monocots, despite taro belonging to Monocot. Recombinant GST–CeCPI fusion protein was overexpressed in Escherichia coli and its inhibitory activity against papain was identified on gelatin/SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. Investigation of its antifungal activity clearly revealed a toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at a concentration of 80 g recombinant CeCPI/ ml. Moreover, mycelium growth was completely inhibited and the sclerotia lysed at a concentration of 150–200 g/ml. Further studies have demonstrated that recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium.     

Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds

Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

OsLOL1, a C2C2‐type zinc finger protein,interacts with OsbZIP58 to promote seed germination through the modulation of gibberellin biosynthesis in Oryza sativa

Seed germination is a key developmental process in the plant life cycle that is influenced by various environmental cues and phytohormones through gene expression and a series of metabolism pathways. In the present study, we investigated a C2C2‐type finger protein, OsLOL1, which promotes gibberellin (GA) biosynthesis and affects seed germination in Oryza sativa (rice). We used OsLOL1 antisense and sense transgenic lines to explore OsLOL1 functions. Seed germination timing in antisense plants was restored to wild type when exogenous GA₃ was applied. The reduced expression of the GA biosynthesis gene OsKO2 and the accumulation of ent‐kaurene were observed during germination in antisense plants. Based on yeast two‐hybrid and firefly luciferase complementation analyses, OsLOL1 interacted with the basic leucine zipper protein OsbZIP58. The results from electrophoretic mobility shift and dual‐luciferase reporter assays showed that OsbZIP58 binds the G‐box cis‐element of the OsKO2 promoter and activates LUC reporter gene expression, and that interaction between OsLOL1 and OsbZIP58 activates OsKO2 gene expression. In addition, OsLOL1 decreased SOD1 gene expression and accelerated programmed cell death (PCD) in the aleurone layer of rice grains. These findings demonstrate that the interaction between OsLOL1 and OsbZIP58 influences GA biosynthesis through the activation of OsKO2 via OsbZIP58, thereby stimulating aleurone PCD and seed germination.     

The families of papain- and legumain-like cysteine proteinases from embryonic axes and cotyledons of Vicia seeds: developmental patterns,intracellular localization and functions in globulin proteolysis

Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V. sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (VPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.     

A major cysteine proteinase, EPB, in germinating barley seeds: structure of two intronless genes and regulation of expression

The barley cysteine proteinase B (EPB) is the main protease responsible for the degradation of endosperm storage proteins providing nitrogenous nutrients to support the growth of young seedlings. The expression of this enzyme is induced in the germinating seeds by the phytohormone, gibberellin, and suppressed by another phytohormone, abscisic acid. In situ hybridization experiments indicate that EPB is expressed in the scutellar epithelium within 24 h of seed germination, but the aleurone tissue surrounding the starchy endosperm eventually becomes the main tissue expressing this enzyme. The EPB gene family of barley consists of two very similar genes, EPB1 and EPB2, both of which have been mapped to chromosome 3. The sequences of EPB1 and EPB2 match with the two previously published cDNA clones indicating that both genes are expressed. Interestingly, neither of these genes contain any introns, a rare phenomenon in which all members of a small gene family are active intronless genes. Sequence comparison indicates that the barley EPB family can be classified as cathepsin L-like endopeptidases and is most closely related to two legume cysteine proteinases (Phaseolus vulgaris EP-C1 and Vigna mungo SHEP) which are also involved in seed storage protein degradation. The promoters of EPB1 and EPB2 have been linked to the coding sequence of a reporter gene, GUS, encoding -glucuronidase, and introduced into barley aleurone cells using the particle bombardment method. Transient expression studies indicate that EPB promoters are sufficient to confer the hormonal regulation of these genes.     

The Squash Aspartic Proteinase Inhibitor SQAPI Is Widely Present in the Cucurbitales, Comprises a Small Multigene Family, and Is a Member of the Phytocystatin Family

The squash (Cucurbita maxima) phloem exudate-expressed aspartic proteinase inhibitor (SQAPI) is a novel aspartic acid proteinase inhibitor, constituting a fifth family of aspartic proteinase inhibitors. However, a comparison of the SQAPI sequence to the phytocystatin (a cysteine proteinase inhibitor) family sequences showed ∼30% identity. Modeling SQAPI onto the structure of oryzacystatin gave an excellent fit; regions identified as proteinase binding loops in cystatin coincided with regions of SQAPI identified as hypervariable, and tryptophan fluorescence changes were also consistent with a cystatin structure. We show that SQAPI exists as a small gene family. Characterization of mRNA and clone walking of genomic DNA (gDNA) produced 10 different but highly homologous SQAPI genes from Cucurbita maxima and the small family size was confirmed by Southern blotting, where evidence for at least five loci was obtained. Using primers designed from squash sequences, PCR of gDNA showed the presence of SQAPI genes in other members of the Cucurbitaceae and in representative members of Coriariaceae, Corynocarpaceae, and Begoniaceae. Thus, at least four of seven families of the order Cucurbitales possess member species with SQAPI genes, covering ∼99% of the species in this order. A phylogenetic analysis of these Cucurbitales SQAPI genes indicated not only that SQAPI was present in the Cucurbitales ancestor but also that gene duplication has occurred during evolution of the order. Phytocystatins are widespread throughout the plant kingdom, suggesting that SQAPI has evolved recently from a phytocystatin ancestor. This appears to be the first instance of a cystatin being recruited as a proteinase inhibitor of another proteinase family. [Reviewing Editor: Dr. Antony Dean]     

Potato hexokinase 2 complements transgenic Arabidopsis plants deficient in hexokinase 1 but does not play a key role in tuber carbohydrate metabolism

Potato plants (Solanum tuberosum L. cv. Désirée) transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 2 exhibited altered enzyme activities and expression of hexokinase 2 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed an 11-fold variation in leaf (from 48% of the wild-type activity in antisense transformants to 446% activity in sense transformants) and an 8-fold variation in developing tubers (from 35% of the wild-type activity in antisense transformants to 212% activity in sense transformants). Despite the wide range of hexokinase activities, no substantial change was found in the fresh weight yield, starch, sugar and metabolite levels of transgenic tubers. However, both potato hexokinases 1 and 2 were able to complement the hyposensitivity of antisense hexokinase 1 Arabidopsis transgenic plants to glucose. In an in vitro bioassay of seed germination in a medium with high glucose levels, double transformants showed the same sensitivity to glucose as that of the wild-type ecotype, displaying a stunted phenotype in hypocotyls, cotyledons and roots.     

Nurse‐plant effects on the seed biology and germination of desert annuals

Nurse‐plants generally have positive effects on understorey species by creating more suitable conditions for stress‐intolerant plants relative to open micro‐habitats. However, long‐term effects of this plant–plant facilitation system have been rarely examined. Seeds of five desert annual species from Atiquipa coastal desert in Southern Peru were used to examine whether different microenvironmental conditions under the nurse‐plants Caesalpinia spinosa Molina (Kuntze) lead to differences in seed biology and germinability of annual plants relative to open, canopy‐free conditions. Seeds collected from plants associated with nurse‐plants were predicted to be (i) larger due to more favourable growing conditions, (ii) more viable and with greater germination rates, (iii) less variable in size and viability due to reduced environmental heterogeneity, and (iv) to germinate faster to avoid apparent competition with other annuals. Seed attribute measurements and germination trials in growth chambers were used to test these predictions. Although the plant abundance of only 2 of 5 species was strongly facilitated by the nurse‐plant, no significant differences were found in seed mass, viability or relative variability between understorey and open micro‐habitats for any of the species. Contrary to our predictions, final seed germination rates of seeds from open micro‐habitats were higher, and the open micro‐habitat treatment was more favourable for germination of seeds from both open and understorey environments. Taken together, these results suggest that plant–plant facilitation does not necessarily affect seed biology traits. Further studies addressing larger distribution ranges and/or density gradients of understorey species will illuminate the potential evolutionary effects of nurse‐plants.     

Seed germination,plant growth and physiological responses of Salsola ikonnikovii to short-term NaCl stress

Salsola ikonnikovii (Chenopodiaceae), a drought-tolerant plant species that is distributed in sand or light-saline soil in Xinjiang, China, produces seeds (fruits) with attached winged perianths. To study the role of the wing in seed germination under salt stress and to further investigate the growth and physiological responses of the plants to salt stress, the germination behaviour of S. ikonnikovii was determined after winged and non-winged seeds were treated with 0–1000 mmol · L^?1 NaCl. Several parameters of two-month old plants that had been treated with NaCl for three weeks were measured. The results revealed that the winged perianths limited germination but protected the seeds from salt damage. The growth of the plants was stimulated by lower concentrations of salt (≤100 mmol · L^?1 NaCl), while increasing salt concentrations inhibited growth. The level of reactive oxygen species and malondialdehyde increased significantly at high concentrations of salt. Correspondingly, concentrations of the osmolytes proline, betaine, and soluble sugars, and the activities of antioxidative enzymes (catalase, peroxidase and superoxide dismutase) increased, but the levels of non-enzymatic antioxidants (carotenoids, glutathione) were significantly reduced at high salt concentrations. These results imply that osmotic adjustment and the antioxidative system may work synergistically to ensure that a plant grows normally under high salt concentrations.     

Does endophyte symbiosis resist allelopathic effects of an invasive plant in degraded grassland?

Allelopathic effects and plant associated systemic endophytic fungi are often thought to play a role in the invasion of exotic plant species. Here, we tested the inhibitory effects of the aqueous extracts of the hemiparasitic weed Pedicularis kansuensis on seed germination and seedling growth of endophyte-free (E−) and -infected (E+) grass species, Stipa purpurea and Elymus tangutorum. The weed extracts significantly inhibited both seed germination and seedling growth of the target grass species. Extracts from the inflorescences gave greater inhibition than those from the stems or roots, while the concentration of the extract had a direct effect on the extent of inhibition. The E+ target plants were less susceptible to the extracts than their E-counterparts. Our results suggest that the allelopathic potential of P. kansuensis will lead to increased frequencies of endophyte infected plants in grass populations.     

Isolation and characterization of a homologous to lipase gene from Brassica napus

Lipase is an important lipolytic enzyme involved in plant lipid metabolism. To analyze its function and roles during seed germination and growth, a full-length cDNA encoding a homologous to lipase gene named BnLIP1 was cloned from Brassica napus, cv. Huyou 15, by rapid amplification of cDNA ends. The BnLIP1 gene had a total length of 1318 bp, with an open reading frame of 1170 bp encoding 389 amino acid residues. Sequence analysis revealed that BnLIP1 protein belonged to the GDSL family of serine esterases/lipases. In B. napus genome, BnLIP1 is represented by several copies with the length of 1601 bp, the gene comprises five exons and four introns. RT-PCR analysis indicated that BnLIP1 showed no tissue-specific expression during reproductive growth and is strongly expressed during seed germination. No expression could be detected until three days after germination, and its peak was registered at the fifth day after germination. In conclusion, BnLIP1-encoded protein is predicted to be a lipolytic enzyme widely expressed at various stages of oilseed rape germination and development. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 410–417. The text was submitted by the authors in English.     

Host-synthesized cysteine protease-specific inhibitor disrupts <Emphasis Type="Italic">Cuscuta campestris</Emphasis> parasitism in tomato

Dodder (Cuscuta campestris) is one of the most important pests of tomato causing severe losses in yield. Cuscutain is a pre-pro-protein produced by dodder that has a cysteine proteinase function essential for normal development of the haustoria and parasitism, which involves the secretion and activation of cuscutain cysteine protease in the host plant tissue. The propeptide subunit of this enzyme has an inhibitory function and restricts the enzymatic activity of cuscutain. Here, we transformed the inhibitory propeptide segment of this enzyme into tomato and examined the tomato resistance to C. campestris. We demonstrate the expression of inhibitory propeptide in transgenic plants and find that it effectively interrupted cuscutain enzyme activity and haustoria development at the endophytic stage. Mature haustoria infecting transgenic hosts showed defects in searching hyphae development and these structures were not elongate, and in most cases no functional haustoria were formed due to inhibitor expression in the transgenic plants after prehaustoria contact. Dodder grown on transgenic lines showed an overall reduction in vigor and fecundity due to defective attachment of haustoria. The increased growth of dodder-challenged transgenic plants relative to controls, demonstrates the efficacy of cysteine protease inhibition in parasite plant control.