MXPA01008158A - Nac1 - Google Patents

Abstract

A novel gene, nacl, has been isolated from Arabidopsis. This gene encodes a protein (NAC1) which has been identified as a member of the NAC family. NAC1 shares a high amino acid sequence homology with other members of the NAC gene products in the N-terminus. Data show that NAC1 belongs to a newly identified family of transcription factors. NAC1 is involved in the regulation of cotyledon and lateral root development. Overexpression of the gene can lead to larger plants with larger roots and more lateral roots than in wild-type plants.

Description

NAC1-ÜN GEN VEGETAL THAT CODIFIES A TRANSCRIPTION FACTOR INVOLVED IN THE DEVELOPMENT OF THE COTILEDON AND OF THE LATERAL ROOT BACKGROUND OF THE INVENTION The NAC genes (including NAM, ATAF1, ATAF2 and CUC2) belong to a relatively large gene family found so far only in plants. These genes encode proteins which are conserved in their N-terminal amino acids but are highly divergent at their C-terminus. Previous genetic studies in petunia (Souer et al., 1996) and Arabidopsis (Aida et al., 1997) have suggested that some members of the NAC family play a role in stem pattern and floral meristem. Petunia embryos that carry the non-apical meristem mutation (nam) fail to develop an apical stem meristem. The occasional stems in the nam shoots have flowers that develop 10 instead of five primordia in the secondary whorl. The double mutants with the green petals homeotic gene show that nam acts independently of the identity of the organ in the whorl 2 and also affects the number of primordia in the whorl 3. Notably, nammRNA accumulates in the cells within the limits of meristem and primordia. It has been shown that nam plays a role in determining the position of the meristems and the primordium (Souer et al., 1996). Mutations in CUC1 and CUC2 (for cup-shaped cotyledons), which are genes of Arabidopsis, cause defects in the separation of cotyledons (embryonic organs), sepals, and stamens (floral organs) as well as in the formation of apical meristems of the stem. These defects are more apparent in the double mutant. The phenotypes of the mutants suggest a common mechanism for separating adjacent organs within the same whorl in both embryos and flowers. The CUC2 gene was cloned and found to encode a protein homologous to the NAM protein of petunia (Aida et al., 1997). The publications and other materials used herein to clarify the background of the invention or to provide additional details regarding the practice, are incorporated by reference, and for convenience are grouped respectively in the attached list of references.

BRIEF DESCRIPTION OF THE INVENTION A new member of the NAC family of Arabidopsis, NAC1, is described. This gene was originally isolated by the ability of its cDNA to alter the cell morphology of the yeast S. pombe when overexpressed when using the method of Xia et al. (nineteen ninety six). The NAC1 analyzes showed that it was expressed in a specific tissue manner with high levels in the root and low levels in the leaves. The total amount in the in situ experiments showed the expression in the actively dividing roots and in the meristems of the stem. NAC1 shares a high sequence homology of amino acids with other members of the NAC gene products in the N-termlnal. In vitro DNA binding studies using a recombinant protein of GST-NAC1 and different truncated derivatives of NAC1 demonstrated an interaction between the -90 region of the 35S promoter and the conserved N-terminal domain of the protein. Interestingly, the yeast assays showed that the C-terminus of NAC1 fused to the DNA binding domain of GAL4 can activate transcription. Analysis of the fusions of different deletions showed that the transactivation domains are located at the C-terminus of the NAC1 protein. In addition, the double-hybrid assays demonstrated that NAC1 can homodimerize and that the dimerization domain is located in the conserved N-terminal region of the protein. A bipartite signal sequence of 21 bp of putative nuclear localization was found in the conserved N-terminal NAC domain. Transgenic Arabidopsis were generated using a GFP-NAC1 fusion construct under the control of a dexametaxone inducible-GVG promoter system (Dex). The analysis of the transgenic lines expressing a GFP-NAC1 fusion protein under the control of Dex induction revealed the nuclear locallzation of the chimeric polypeptide in vivo. These data indicate that NAC1 belongs to a recently identified family of transcription factors. One aspect of the invention is a transgenic plant comprising a gene which encodes NAC1 or a functionally equivalent protein (which means a protein that binds to the same DNA binding site and has no NAC1 and which is at least 70% identical with, preferably 80% identical with, more preferably 90% identical with, and more preferably at least 95% identical with NAC1) which causes the transgenic plant to grow larger than a non-transgenic plant. The plant may be larger because they are heavier, because they have larger leaves, because they have thicker stems, because they have more roots, and / or because of their large roots. A second aspect of the invention is a gene encoding NAC1 or a functionally equivalent protein wherein plants that are transgenic for this gene grow larger than a non-transgenic plant. A third aspect of the invention is NAC1 or a functionally equivalent protein wherein if a plant is made transgenic with a gene or gene encoding NAC1 or said functionally equivalent protein said transgenic plant will grow larger than a non-transgenic plant. Another aspect of the invention is a transgenic plant cell which contains a gene encoding NAC1 or a functionally equivalent protein which causes the plant cells to grow larger than a non-transgenic cell.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Tissue-specific expression of the NAC1 gene. Autoradiography of gel blots of RNA containing 10 ig of total RNA isolated from different tissues, as marked. The shoots were harvested from two-week-old plants. The leaves of cauliferous (leaf), the main or lateral stems (stems), the groups of flowers (flower), and the silicua of different stages were harvested from plants of 35 to 40 days grown in soil. Roots were taken from two-week-old shoots with vertical growth in boxes with SM medium. The filters were hybridized with radiolabeled C-terminal NAC1 and then tested with the 18S radiolabeled rRNA probe as charge control. Figure 2. Total amount of in situ study of NAC1 in shoots and flowers. The total amount of in situ hybridization was carried out on shoots and flowers with a C-terminal specific antisense probe labeled with digoxigenin (DIG) (A, B, C, D and E) and with a DIG-labeled probe ( F and G). A and F are 12-day-old shoots. The root materials are from 12-day-old shoots and D is a 7-day-old shoot. The flowers were taken from the plants that grew for 40 days in soil.

Figure 3. Binding of the GST-NAC1 fusion protein to the -90 region of the CaMV 35S promoter. (A) Affinity of the binding of the GST-NAC1 fusion protein to the -90 regions of the 35S promoter. the amount of proteins is as follows: line 1, 500 ng of GST protein. Lines 2 to 9 are NAC1 fusion proteins. Lines 2 and 6, 10 ng; lines 3 and 7, 100 ng; lines 4 and 8, 250 ng; lines 5 and 9, 400 ng. As the cold competitor, an unlabeled DNA fragment was used. (B) Map of the following deletions from the NAC1 to GST merger. (C) Different deletions of the binding of the NAC1 fusion protein to the -90 region of the 35S promoter of CaMV. 20 ng of each recombinant protein was used. Figure 4. Specific interaction between NAC1 and NAC1 in the yeast double hybrid system. The HF7 yeast cells were co-transformed with the plasmids expressing the GAL4BD alone or the GAL4BD -NAC1 fusion and the plasmids expressing the GAL4AD alone or fused to NAC1. (A) The cells were seeded on dishes with or without histidine plus 10 mM 3-AT (Sigma) according to the distribution shown in the center of the figure. The ability to grow in the absence of histidine depends on the functional reconstitution of GAL4 activity. (B) The map of different deletions of the NAC1 fusion protein in the pGA vector. (C) The ability of interaction between the different versions of NAC1 deletions in the double hybrid system assays. Figure 5. Localization of the transactivation domain in the NAC1 protein. The different deletion versions of NAC1 were cloned into the Gal4 DNA binding vector and transformed into the yeast strain HF7c. The transformation mixture was seeded on MM dishes with histidine or without histidine as a supplement with the necessary amino acids. The results were taken from the dishes that grew for three days. Figure 6. Effect of overexpression of NAC1 in plants. (A) Growth of wild-type plants in the absence of Dex. (B) Growth of wild-type plants in the presence of Dex. (C) Growth of transgenic plants in the absence of Dex. (D) Growth of transgenic plants in the presence of Dex. Figure 7. Changes in cotyledon development in specific antisense plants for C-terminal. The SEM shows the abaxial surface of the cotyledon. A and D (detail) are wild type, B and E (detail) are non-severe phenotypes, C and F, G (detail) are severe phenotypes. The boxes with letters in A, B and C show the regions that are detailed. Figure 8. Seeds of wild type plants or transgenic plants with NAC1 antisense that were germinated in MS or MS medium plus Dex. (A) Wild type in the presence of Dex; (B) Wild type induced with Dex; (C) transgenic in the absence of Dex; and (D) transgenic induced with Dex.

DETAILED DESCRIPTION OF THE INVENTION The nac7 gene was isolated in a project to isolate the proteins related to the plant cytoskeleton, with the cell cycle and with the polarity using Schizosaccharomyces pombe. An A. thaliana cDNA library under the control of the threamine repressible nmtl promoter of pREP5N was transformed into the wild-type S. pombe cells. One transformant showed elongated and multisept cells when the promoter was depressed. A cDNA clone was isolated from this transformant and retransformed in S. pombe to confirm that cell shape changes were due to cDNA expression. The isolated cDNA (At012) encodes a single open reading frame (ORF) of 324 amino acids (SEQ ID NO: 2). The sequence of the putative protein was searched again in GenBank. The BLAST search indicated that the protein is novel. It was found that the N-termini of the protein contains a NAC domain, a domain that is found in members of the NAC gene family (NAM, ATAF1, ATAF2, CUC2). This domain covers the first 175 amino acids of the protein. The rest of the protein encoded by nací does not have a high homology with any known sequence. Homology, for polypeptides, is typically measured using sequence analysis software. See, for example, the Sequence Analysis Software package from Genetics Computer Group, University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wisconsin 53705. Protein analysis software matches similar sequences using homology measurements assigned to several substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine, serine, threonine; lysine, arginine; and phenylalanine, tyrosine. The NAC family of genes has been identified only in plants. These genes encode proteins that are highly conserved at the N-terminus but which vary in the rest of the protein. The first two members, ATAF1 and ATAF2, were found in Arabidopsis and isolated for their ability to activate the 35S cauliflower mosaic virus (CaMV) promoter in yeast (H. Hirt, GenBank accession numbers X74755 and X74756). The SENU5 cDNA, isolated in tomato senescence studies (GenBank accession number Z75524; John et al., 1997), and NAM protein (Souer et al., 1996) the product of the petunia nam gene, they are required for the proper development of the apical meristems of the stem and it has been proposed that they determine the location of the meristem. CUC2 (Aida et al., 1997) is a member of the NAM family and is a homolog of NAM of Arabidopsis. The NAP protein of Arabidopsis is also a member of the family (Sablowski and Meyerowitz, 1998). Finally, GRAB1 (GenBank access number AJ010829) and GRAB2 (GenBank access number AJ010830) are two most recently reported members of the NAC family. These are from wheat and can interact with the wheat dwarf virus RepA protein (WDV). The trans-overexpression of the GRAB1 and GRAB2 genes under the control of the 35S promoter can inhibit the replication of WDV in the culture of wheat cells. The work reported here details that the NAC domain in five blocks is based on the conservation and change of each block. The alignment of the NAC1 protein sequence with all these proteins revealed that NAC1 has similar characteristics with other members of the NAC family which correspond to the recently identified NAC domain. The genomic structure of nací was analyzed by comparing the genomic sequence with the cDNA sequence. The NAC1 gene is located in the chromium. This gene includes two introns in the region encoding the N-terminus. The first intron is 1215 base pairs and is inside of the codon of amino acid 67. The second intron is 102 base pairs and is located between the codons of amino acids 160 and 161. A search of the domain was carried out and led to the identification of a two-part signal of putative nuclear localization within of the NAC1 domain from NAC1. This signal sequence of 21 amino acids is localized from amino acid 117 to 137 and was the first found in a member of the NAC family. Carrying out the same search with the other reported members of the NAC family failed to reveal a similar signal. The NAC homologs were observed in an EST database of rice but no homologs were observed in other organisms such as mammal, Drosophila and yeast.

Regulation of the development of the NAC1 gene To investigate the roles of the NAC1 gene during plant development, its expression pattern was analyzed by Northern analysis using a specific probe for nací. Because members of the NAC family are highly conserved at the N-terminus even at the DNA level, a C-terminal probe was used. 10 μg of the total RNA of each tissue was used for hybridization. A relatively high level of nacre RNA expression was detected in the roots (figure 1). A low level was observed in the two-week-old shoots which contain all plant tissues. Very low expression was detected in the material obtained from stems and leaves. There was no expression detected in the mixed stages of the siliceous and the mixed floral and inflorescence material, even after a large exposure. The total amount of in situ studies of 7-day and 12-day plants were carried out using both sense and antisense probes for the C-terminal coding region of the gene. The expression of nací was observed in the roots that actively divide and in the apical meristems of the stem (figure 2). The expression was especially high in the region of the split root tip (figure 2A and 2B) and in the regions of lateral root formation (figure 2C). A lesser expression was detected in the cotyledon and young leaves. All these results correspond to the Northern results. No clear specific hybridization was detected in the flower (figure 2E). Also, no signal was detected in the siliques and the stems (data not shown). No specific signal was observed in the shoots hybridized with the sense RNA probes (figure 2F and 2G).

DNA binding and DNA binding domains The first two genes of the NAC family, ATAF1 and ATAF2, were cloned for their ability to activate a 35S promoter of Cauliflower Mosaic Virus (CaMV) constructed in yeast. These two proteins, as well as the other genes of the NAC family, are conserved in the N-terminal domain of NAC. The GRAB1 and GRAB2 proteins from wheat also bind to the -500 region of the 35S promoter. The CaMV 35S promoter contains several known DNA binding elements. It has been identified that numerous plant transcriptional factors bind to different elements in the regulatory region of the promoter. To evaluate whether the NAC1 protein can bind to the 35S promoter, the -90 region of the 35S promoter was initially used as a probe, was cloned into the GST fusion vector pGEX-4-2T and transformed into E. coli. The recombinant GST fusion protein was purified and an assay was carried out to test specific binding to DNA. Since the GST fusion protein had bound, GST itself was used as a control. GST has no ability to bind to the -90 region of the 35S promoter even when 500 ng of the purified protein was used (see Figure 3A). Different amounts of the purified protein GSTNAC1 were tested. The binding was detected with as little as 10 ng of protein and with only 4 hours of exposure in the film (Figure 3A). It should be noted that the purified GST protein contained only about 10% of the intact GST fusion form. Most of the protein was degraded during growth and bacterial purification. This is because after IPTG is added to induce the expression of the protein, the bacteria can no longer grow well and then the protein starts its degradation before purification. The addition of an excess of the cold 35S promoter which is a competitor of DNA abolished the formation of the DNA-protein complex in a concentration-dependent manner (Figure 3A). These results demonstrate that the NAC1 protein binds specifically to the -90 region of the 35S promoter. The binding of NAC1 to an AS-1 element and to a mutant form of the element, -83 to -63 of the 35S promoter, was also evaluated. It has also been identified that zygote transcriptional factors bind to this region. The binding affinity was reduced and there was no clear difference between the original AS-1 element and the mutant form. This result indicates that NAC1 has a different DNA binding site than the bZIP transcriptional factors and the sequences around the AS-1 element are important for the specific binding of NAC1. To evaluate the DNA binding domain of NAC1, different forms of naci deletion were cloned into the GST fusion vector and the fusion proteins were isolated to evaluate the DNA binding capacity of the -90 region of the 35S promoter. 20 ng of each protein was used in this assay. The results are shown in Figure 3C. The truncated form of GSTNAC1 contained the first 199 amino acids, of the intact NAC domain. This maintained specific binding similar to the DNA probe. A truncated version containing only the first 3 intact blocks of the NAC domain and part of the IV block lost the ability to bind to DNA. Similarly, upon complementation of this form of deletion, the rest of the protein containing part of the IV block and the intact V block did not bind to the DNA probe. Together these results indicate that DNA binding domains are located in the NAC domain and that block IV is important for DNA binding and block V is not sufficient for DNA binding.

NAC1 Homodimers and the dimerization domain Transcriptional factors usually form dimers where they bind to DNA. To determine if NAC1 forms dimers, the inventors used a double hybrid technique. The method was used efficiently to evaluate dimerization and to study the protein-protein interaction including dimerization. The intact NAC1 was fused to both the Gal4 DNA activation domain and the DNA binding domain and was co-transformed into the yeast with the reporter gene. A clear interaction was observed when NAC1 was expressed by both vectors. The yeast was able to grow in three days even when 10 mM 3-AT was applied to block non-specific interactions (Figures 4A). It should be mentioned that when expressed only in the DNA binding vector it can autoactivate the system at a low level so that the yeast can grow very slowly (Figure 4A). To identify the region of NAC1 required for dimerization, the inventors constructed a series of deletions and analyzed their ability to form complexes in yeast. First a C-terminal deletion was made from amino acid 200 to the end of the protein. This left a protein of the first 199 amino acids. This fragment of the 199 amino acid protein contained the intact NAC domain and was able to form dimers with the intact NAC1. This version of 199 amino acids did not give a background signal similar to the NACI version of wild type total length. Therefore the 199 amino acid version was used as the fixed participant to evaluate other deletions. A variety of different deletions were prepared in the vector with the activation domain (Figure 4B). When the same deletion (absence of the sequence beyond amino acid 199) was prepared in pGA the interaction was as strong as with the intact protein. But when the deletion affected block IV of the NAC domain and the construct contained only the first three intact blocks of the NAC domain, the formation of the dimers was abolished (Figure 4C). The deletion of the N-terminal left only the back half of block IV and the intact block V resulted in a protein incapable of forming dimers. The data strongly support the conclusion that the NAM domain of NAC1 contains the dimerization domain. Block IV is important for dimer formation but block V itself is not sufficient to form a dimer. The NAC1 protein can form homodimers and this was confirmed by gel filtration. When the bacterium expresses a protein of the first 199 amino acids of NAC1, the purified peptide including the complete NAC domain ran as twice the size in both a native gel and a denaturing gel.

Transactivation and transactivation domain In the yeast double hybrid assays the inventors found that NAC1 can auto-activate the system at a low level but the C-terminal deletion can not do so. Therefore the inventors used a single-hybrid method modified with the hybrid transcription factor to identify the activation domain of the NAC1 gene. The pGBT8 vector was used, which contains only the Gal4 DNA binding domain but not the activation domain. The different deletions of the NAC1 gene were cloned into this plasmid in a fusion of the Gal4 DNA binding domain and the resulting plasmid was transformed into yeast containing the reporter gene UASQa-CYC1-HIS3. The selection was carried out on the dishes without histidine. If the part of the fusion protein contained the activation activity, the Gal4 transcript had to be reconstructed and activated the HIS3 promoter. With the synthesis of histidine, the yeast can grow on medium without histidine. The Gal4 activation domain was used as the positive control and this gave total activity as shown in Figure 5. As mentioned above, the complete NAC1 protein gave only low activation activity. The N-terminal fusion containing the first 132 amino acids of the first 199 amino acids gave no activity. Similarly, a fusion with a peptide fragment of amino acids 143-199 gave no activity. In contrast, a fusion containing amino acids 133 to the carboxyl terminus completely activated the reporter gene giving a signal as strong as the positive control of the transcription factor Gal4. The use of a fusion containing only amino acids 143-269 also gave total activity. This is a highly acidic region of the amino acid. The C-terminal fusion containing only the last 54 amino acids resulted in low activity. The results indicate that the activation domain of NAC1 is located at the C-terminus. Because the NAC family has been observed only in the plants and not in other organisms, it was important to check the transactivation activity in plants. An in vivo experiment was designed to check the activation domain. Two plasmids were constructed. The first one carried the DNA binding cassette of Gal4 / MCS-Nos terminal labeled with a 35S promoter of CaMV. The evaluated gene can be cloned into MCS in reading frame with the DNA binding domain of Gal4. The other plasmid contained the construction of the reporter gene 6xUAS-TATA-Luc which can express luciferase if some proteins can bind to the 6xUAS element to activate the promoter. The co-bombardment with the two plasmids together in different combinations demonstrates that the system is reproducible and applicable. The inventors used the activation domain for the VP16 virus as a positive control and this gave a very high activity. In contrast, neither the second plasmid (pTALuc) alone nor this plus the pGal vector gave significant luciferase activity. A similar result was obtained as that for the yeast system. Activation of full transactivation was identified in amino acid peptide 143-269. The last 54 amino acids also gave low activity. The N-terminus of NAC1 did not give activity nor did the control vector. In both yeast systems and plants, it has been shown that NAC1 contains a transactivation domain and the region corresponding to this activity is between amino acids 142-269.TRANSGENIC ARABIDOPSIS PLANTS THAT OVEREXPRESS AND SUBEXPRESAN NAC1 In this study, a glucocorticoid-inducible transgenic system was used as previously described (Aoyama and Chua, 1997). A transcription factor regulated by glucocorticoid is encoded by the GVG whose transcription is controlled by the 35S promoter of CaMV. Transcription of the transgene is controlled by the glucocorticoid-activated promoter (6XUASgai4) - The cDNA encoding the complete 1287 base pair NAC1 (SEQ ID NO: 1) was cloned into the binary plant transformation vector pTA7002 in both directions and transformed separately into the Arabidopsis thaliana lansberg ecotype by the root transformation method. The transgenic lines that overexpressed and underexpressed were selected from media containing hygromycin. Six independent transgenic lines were selected from those that overexpressed and four independent lines from those that underexpressed. All lines segregated for one of the DNA-T locus according to the segregation ratios were resistant to hygromycin. The six transgenic plants that overexpressed were of three different phenotypes. Four of the lines were of one phenotype while the two remaining lines each showed a different phenotype. These last two phenotypes are similar to antisense plants and are described below. Studies of the first four lines of similar phenotype showed that those plants grow more rapidly and grow larger than non-transgenic plants both under in vitro conditions and in soil growth. The most lateral roots were produced when induction conditions such as growths in vertical boxes or in plant containers were used. The data are presented for in vitro growth conditions. Sprouts of different ages were transferred from MS medium to MS medium and MS medium plus 10 μM dexamethasone (Dex), and then checked after one week of growth. Ten plants of each homozygous line were weighed after cleaning the agar or the soil of the plants. The one-week-old shoots that were transferred to Dex medium were 1.1 to 1.3 times heavier than the non-induced plants, the 15-day-old shoots were 1.3 to 1.8 times heavier, and the 25-day-old shoots were 1.4 to 2.6 times heavier. The increased weight of induced plants resulted from larger leaves, thicker stems and more roots, with leaf size being the largest contributor. Leaf epidermal cells from different parts of the plants were analyzed by scanning electron microscopy (SEM). The SEM showed that the cell size of the old leaves was greater than the size of the control cell while there was no clear difference in cell size in the newly growing leaves. The roots that grew vertically from a representative line are shown in figure six. The densest and largest lateral roots occurred in the lines induced with Dex. Total RNA was prepared from induced plants for 48 hours and controls and were measured via Northern blots assayed with the specific C-terminal probe for nací. The results indicate that all four transgenic lines show that the transgene is expressed. The size of the transgene mRNA is larger than the size of the mRNA and is easily distinguishable. The two lines of overexpression remaining to themselves as the four lines selected for the transgenic antisense plants showed that they share some different combinations of phenotypes. One of the two overexpressing lines and one of the antisense lines showed the curved cotyledon phenotype in the shoot stage. No clear phenotypes were detected in the late stage of plant development of the inflorescence. Two antisense lines showed very typical phenotypes in the shoot stage as well as the cuc2 mutant, in this case, the single cotyledon, heart-shaped and fused and more severe, the rosette shoots were detected at a low percentage around 7-10% of germination shoots. Most of the shoots, which can not produce the meristem of the apical stem, died later. One of the antisense lines and one overexpression line did not appear in any way different from the non-induced plants during all stages of shoot and vegetative growth. But at the stage of inflorescence development, the floral organs, particularly petals and stamens, were affected. The phenotypes found in the first flowers of the inflorescence. Those flowers that had short petals and stamens, and those with the most severe phenotype where the flower could not be opened at all. Some of them could be opened but the short stamens could be observed. Those flowers were sterile males and females. This type of phenotype overcomes the overexpression of another member of the NAC family of Arabidopsis - the NAP gene.

SU3EXPRESSION OF THE NAC1 SPECIFICIAL C-TERMINAL GENE IN THE TRANSGENIC PLANTS OF ARABIDOPSIS Since members of the NAC gene family are highly conserved at the N-terminus, not only at the amino acid level but also at the level of the nucleotide sequence, antisense nucleic acids for this region will affect homologous genes as shown in petunia . In addition, C-terminal specific antisense constructs were prepared using vector pTA7002 and transformed into Arabidopsis as described above. For this construction, the inventors used the C-terminal fragment digested with Bam \ -Not \ 0.6 kb. Northern blots developed only one band corresponding to the NAC1 gene in all tissues when this fragment was used as a probe for the four lines of the six homozygous lines that were selected for analysis. All show similar phenotypes, mainly the affected cotyledon and the development of the root. No clear phenotypes were detected during all stages of plant growth. A representative line of C-specific antisense plants is described herein. Wild-type cotyledons or non-induced suckers showed a light curve and smooth surface. In the first week of germination a clear phenotype was not detected in the development of the cotyledon but in the shoots of ten days of age, 20 to 25% showed phenotype of severe curve of the cotyledon. A few also showed a receding curve phenotype. The phenomenon can be easily detected under light microscopy or can even be observed directly. In the later stage, black spots can be observed on the surface of the cotyledons. The abaxical epidermal cells were checked by SEM and abnormal cell expansion was found on the surface of all the cotyledons, although different levels of severity were observed (figure 7). Those cells lost the normal puzzle-like structure and became disordered with different cell sizes and cell shapes (Figure 7C, 7F and 7G). The cotyledons of those 75 to 80% of shoots that did not show a clear or severe curve under light microscopy were also checked by SEM. The results of SEM indicated that the cell surface of those cotyledons were also abnormal. The cells looked swollen and more expanded. The phenotypes of the root were easily detected in shoots with vertical growth. The seeds were germinated on MS or MS medium plus Dex. After a week of gemination, the shoots were transferred to the same fresh medium and continually grown for a week and then checked. A few roots were produced in the shoots containing C-terminal antisense expression. Three lines had short or some lateral roots and one line had almost no lateral roots. Those shoots that had short or few lateral roots were checked by microscopy and it was found that the lateral roots could be started but could not be elongated. Therefore the shoots looked as if they contained few roots compared to the non-induced shoots (Figure 8). Similar results were obtained by C-terminal antisense plants grown in liquid. The one-week-old shoots were added to liquid medium MS or MS plus Dex and after 12 days of growth the non-induced roots are longer than the roots induced from the shoots. The present invention is further detailed in the following examples, which are offered by way of illustration and are not intended to limit the invention in any way. Standard techniques well known in the art or techniques specifically described below were used.

EXAMPLE 1 Plant materials and growth conditions The ecotype Arabidopsis thaliana lansberg was used for the experiments. Seeds were sterilized on their surface with 20% bleach plus 0.01% Triton X-100 and washed three times in sterile water. After the first wash, 0.15% was added to the agarose seed and then Dexametasone (Dex) at different concentrations with 3% sucrose was planted on MS or MS medium. Plates were incubated at 4 ° C for two days and transferred to a tissue culture room at 22 ° C under long day conditions (16 hours light / 8 hours dark). After two to three weeks, the shoots were sown on land and grown in a growth chamber with a photoperiod of 15 hours of light / 8 hours of darkness at 22 ° C and 75% humidity. For Dex treatment, dexamethasone (Sigma) was dissolved in DMSO to make a storage solution of dexamethasone 100mM which was stored at -20 ° C. For in vitro growth plants, Dex was added to dishes 0.1, 1 and 10 iM. The Dex was added continuously to the medium once a week. Light concentrators and plant concentrators were used. Dex was initially dissolved in sterile water at 1/25 volume of medium and then added to the surface of the medium. For the treatment of plants grown in soil, Dex was added to sterile water containing 0.01% Triton X-100 at 30 μM and then sprayed once every two days to cover all plant surfaces. For controls, sterile water containing 0.01% Triton X-100 was sprayed on the same number of plants.

EXAMPLE 2 Transformation and plant transformation constructions The binary transformation plasmid pTA7002 containing the complete two-component system inducible with giucocorticoid (Aoyama and Chua, 1997) was used as a vector. For the overexpression and complete antisense constructs, a 1287 base pair Sall-Nori fragment containing the cDNA encoding complete NAC1 was blunt-ended and cloned into the pTA7002 vector treated with goat intestinal alkaline phosphatase and digested with Xhol. To prepare a C-terminal specific antisense construct, the 605 base pair BamHI-Notl fragment containing the specific C-terminal region of cDNA encoding NAC1 was blunt-ended and cloned into the vector pTA7002 as mentioned above . For the transgenic constructs containing the GFP-NAC1 fusion cassette, a Nael-Sall fragment was generated by PCR containing just the coding region of NAC1 and cloned into a GFP fusion vector pGFP2 (GA) 5ll intermediate digested with A / ael and I went out to produce a pGFPNAd plasmid. Then the GFPNAC1 fusion was cleaved by Xbal plus Pací and cloned into the pTA7002 vector using the same procedure as described above. The roots of Arabidopsis thaliana lansberg were used as the plant material for transformation (Valvekens et al., 1988). The T2 seeds were germinated on SM plates containing 20 ig / mL of hygromycin B to select the resistant plants and transfer them to soil to generate and obtain the homozygous T3 seeds. two independent lines of homozygous T4 plants were used with a single insert from each construction for detailed analysis.

EXAMPLES 3 Northern analysis and total amount of In Situ analysis Total RNA was isolated from different tissues when using the Qiagen RNA preparation kit. Northern blot analyzes were carried out according to Nagy et al. (1988). For the probes, a PCR fragment spanning amino acids 200 to 324 was labeled with á-32P dCTP using a Redi-pirmed tagging device (Amersham International). Total plants or flowers were set for the total amount of in situ hybridizations. Antisense RNA for the specific C-terminal region of NAC1 was labeled with digoxigenin UTP (Boehringer Biochemica, Mannheim, Germany). Hybridization was detected using the anti-digoxigenin Fab fragment conjugated with alkaline phosphatase. As control probes, sense transcripts were prepared from the same clone using the T3 transcriptase.

EXAMPLE 4 Yeast Double Hybrid Analysis The double hybrid analyzes (Bartel et al., 1993, Fields and Song, 1989, Chevray and Nathans, 1992, Lee et al., 1995) were applied to the dimerization analyzes. The yeast strain HF7c (MATa ura3-52 his3-200 ade2-101 Iys2-801 trpl-901 Ieu2-3,112 gal4-542 gal 80-538 LYS2 :: GAL1? AS-GAL1TATA ~ HIS3 URA3 :: GAL417mars (? 3) -CyC1tATA-LacZ which contains the two reporter genes LacZ and HIS3.The cotransformation of yeast with the plasmids carrying the different binding domains of the Gal4 DNA and the Gal4 activation domain of fusion were carried out by the methods known to those skilled in the art, see, for example, Burke and Olson, 1986; Rudolph et al., 1985; Sakai et al., 1984. Other conditions have been described in detail elsewhere and are well known to those skilled in the art. To corroborate the interaction between the fusion proteins, an α-galactosidase activity was evaluated by a replicate filter assay. The two versions of NAC1 were cloned into the vector pGBT8, of the DNA binding domain of Gal4, a derivative of pGBT9 (Clontech) which contains a more versatile polyadaptator. Plasmid pGBNAC11-324 contains the complete open reading frame for NAC1 fused to the binding domain of Gal4 DNA. The plasmid pGBNAC11-199 only contain the nucleic acid encoding the first 199 amino acids of the same vector. Different truncated versions of NAC1 were fused to vector pGAD424 of the Gal4 activation domain. Plasmid pGANAC11-324 contains the complete open reading frame for NAC1. pGANAC11-199 contains the nucleic acids encoding the first 199 amino acids of NAC1. The plasmid pGANAC11-142 contains the nucleic acids encoding the first 142 amino acids of NAC1 and the plasmid pGANAC 1143-324 contains the nucleic acids encoding the amino acids from 143 to the C-terminal end of the peptide.

EXAMPLE 5 Transfection of epidermal cells by particle bombardment The petals were cut into 2x2 cm pieces and put on filters soaked with MS liquid medium. 2 μg of each plasmid combination was used for each shot in a 27-inch mercury vacuum using a helium pressure of 1100 psi. Then the bombarded dishes were incubated in the plant growth room overnight (16 hours) and 50 mM Luciferin (Promega) was sprayed onto the material. The results were checked after 10 minutes of incubation.

EXAMPLE 6 Production of GST fusion proteins and DNA binding The PCR product containing the entire coding region of the NAC1 gene was cloned into pGEX-4-2T to produce the fusion construct pGST-NAC1. GST-NAC1 1-199 was generated by digestion of pGST-NAC1 with Bam I and San and was treated with Klenow and religated to produce pGST-NAC1 1-199 which contained only the first 199 amino acids of the NAC1 protein. GST-NAC1 1-132 was obtained by cutting pGST-NAC1 with Xhol plus Sali and relinking it. GST-NAC1 133-324 was constructed by cloning an Xhol-Not fragment from the original cDNA clone into pGEX-4-2T. The plasmids were transformed into E. coli BL21 (DE3). The transformants were grown at OD600 from 0.6 to 0.9 and then induced to express the fusion protein at 30 ° C for 4 hours by the addition of IPTG at 0.4 mM. The cells were washed once with PBS and resuspended in GST buffer (GCB: 50 mM Tris-HCl at pH 8.0, 200 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM α-mercaptoethanol, 5 μg / ml of each of leupeptin, pepstatin and aprotinin, and 1 mM PMSF). The cells were lysed by sonication on ice (5x30 seconds), and the lysates were clarified by centrifugation (15 minutes at 12,000 rpm). The fusion protein was recovered by glutathione-sepharose bed affinity chromatography (Pharmacia). The DNA binding reaction contained 2x105 cpm of the DNA probe, 5L of 4x of the reaction buffer (100mM NaCl, 40mM HEPES, pH 7.5, 2mM EDTA, 20% glycerol, 140mM α-mercaptoethanol) , 0.5 μg poly IDC (Pharmacia), 1 μL of Nonidet P40 1% and different amounts of proteins in a total volume of 20 μL. 10 μl of the reaction was used on a 4-6% native gel with 1/2 TBE. A 100 base pair SamHI fragment containing 35S-90 from the region was labeled by α-32 PdCTP in a Klenow labeling reaction. Details of the preferred embodiments of the invention, it is understood that the description is intended to be illustrative rather than in a limiting sense, and it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and scope. of the appended claims.

List of references Aida M, et al. (1997). The plant Cell 9: 841 -857. Aoyama T and Chua N-H. (1997). Plant J. 11: 605-612. Bartel PL, et al. (1993). "Using the 2-hybrid system to detect protein-protein interactions." In Cellular Interactions in Development: A Practical Approach, Oxford University Press, pp. 153-179. Burke DT and Olson MV (1986). DNA 5: 325-332. Chevray PM and Nathans DN (1992). Proc. Nati Acad. Sci. USA 89: 5789-5793.

Fields S and Song O-K (1989). Nature 340: 245-246. John I, et al. (1997). Plant Mol. Biol. 33: 641-651. Lee JE, et al. (nineteen ninety five). Science 268: 836-844. Nagy F, et al. (1998). In Plant Molecular Biology Manual, B4 (Gelvin S and Shilperoort R, eds). Dordrecht, The Netherlands; Kluwer Academic Publishers, pp. 1-12. Rudolph H, et al. (1985). Gene 36: 87-95. Sablowski RWM and Meyerowitz EM (1998). ' Cell 92: 93-103. Sakai K, et al. (1984). Mol. Cell. Biol. 4: 651-656. Souer E, et al. (nineteen ninety six). Cell 85: 159-170. Valvekens D, et al. (1998). Proc, Nati. Acad. Sci. USA 85: 5536-5540. Xia Q, et al. (nineteen ninety six). Plant J. 10: 761-769.

SEQUENCE LIST < 110 > Xie, Qi Ch.ua, Nam-Hai Institute of Molecular Agrobiology, The National University of Singapor. < 120 > NACÍ - A VEGETAL GENE THAT CODIFIES A TRANSCRIPTION FACTOR INVOLVED IN THE DEVELOPMENT OF THE COTILEDON AND THE SIDE ROOT < 130 > 2248-115 < 140 > Not yet assigned < 141 > 1999-02-11 < 160 > 2 < 170 > Patent in Ver. 2.0 < 210 > 1 < 211 > 1287 < 212 > DNA < 213 > Arabidopsis thaliana < 220 > < 221 > CDS < 222 > (89) .. (1060) < 400 > 1 gtcgaccacg cctccgtctt tatctctctt ttcctcttaa ccatccacta atcaaacact 60 aaaacctaga aaaaaaaagg atcaaatc atg gag ac ga gaa gag atg aag 112 Met Glu Thr Glu Glu Glu Met Lys. 1 5 gaa agt agt ata age atg gtg gag gca aag ttg cct cg gga ttc aga 160 Glu Ser Ser lie Met Val Glu Ala Lys Leu Pro Pro Gly Phe Arg 10 15 20 ttt falls ccg aag gac gat gag ctt gtc tgc gat tac ttg atg aga cga 208 Phe His Pro Lys Asp Asp Glu Leu Val Cys Asp Tyr Leu Met Arg Arg 30 35 40 tcg ctt cac aat aat cat cga cea cct ctt ctt gtc ctg atc ca gtc gat 256 Ser Leu His Asn Asn His Arg Pro Pro Leu Val Leu He Gln Val Asp 45 50 55 ctc aac aag tgt gag cct tgg gac atc cea aaa atg gca tgc gtg gga 304 Leu Asn Lys Cys Glu Pro Trp Asp He Pro Lys Met Wing Cys Val Gly 60 65 70 ggg aag gat tgg tat ttc tac age caga aga gac cga aaa tac gcg acg 352 Gly Lys Asp Trp Tyr Phe Tyr Ser Gln Arg Asp Arg Lys Tyr Wing Thr 75 80 85 ggg ctg aga act aac cga gca acg gcc acc gga tat tgg aaa gcc acc 400 Gly Leu Arg Thr Asn Arg Wing Thr Wing Thr Gly Tyr Trp Lys Wing Thr 90 95 100 ggc aaa gac aga acc att cta aga aga ggt aag cta gtt ggg atg agg 448 Gly Lys Asp Arg Thr He Leu Arg Lys Gly Lys Leu Val Gly Met Arg 105 110 115 120 aag here ttg gtt ttc tat ca ggt ggt cg cct cg cg ggc cgt aaa acc 496 Lye Thr Leu Val Phe Tyr Gln Gly Arg Wing Pro Arg Gly Arg Lys Thr 125 130 135 gat tgg gtc atg cac gaa ttc cgt ctc ca gga tet cat cat cct ccc 544 Asp Trp Val Met His Glu Phe Arg Leu Gln Gly Ser His Hi s Pro Pro 140 145 150 aat cat tet ctg age tet cea aag gaa gac tgg gtc ttg tgt agg gta 592 Asn Leu Ser His Ser Ser Pro Trp Asp Lys Glu Val Leu Cys Arg Val 155 160 165 ttc cat aag aat acg gaa aga gga gtt tgt ata gac aac atg gga age 640 Phe His Lys Asn Thr Glu Gly Val He Cys Arg Asp Asn Met Gly Ser 170 175 180 tgt ttt gat gag here gcc tet gca tcg ctt cct cea ctg atg gat cct 688 Cys Phe Asp Glu Thr Ala Ser Ala Ser Leu Pro Pro Leu Met Aep Pro 185 190 195 200 tac atc aac ttt gac caa gaa ccc tet tet tat ctc agt gat gat cat 736 Tyr He Asn Phe Asp Gln Glu Pro Ser Ser Tyr Leu Ser Asp Asp His 205 210 215 cac tac atc aat gag cac gta ccc tgc ttc tcc aat ttg tea cag 784 His Tyr He He Asn Glu His Val Pro Cys Phe Ser Asn Leu Ser Gln 220 225 230 aac caa acc tta aac tcg aac cta acc aac tea gtc tet gaa ctc aag 832 Asn Gln Thr Leu Asn Ser Asn Leu Thr Asn Ser Val Ser Glu Leu Lys 235 240 245 att cea tgc aag aac cct aac ccc ttg ttt act ggt ggt tea gcc tea 880 He Pro Cys Lys Asn Pro Asn Pro Leu Phe Thr Gly Gly Ser Wing Ser 250 255 260 gcc acg ctc here ggc ctc gac tea ttc tgt tet tea gat cag atg gtt 928 Wing Thr Leu Thr Gly Leu Asp Being Phe Cys Ser Being Asp Gln Met Val 265 270 275 280 ctc aga gct cta ctc agt cag ctc act aag att gga age ctc ggg 976 Leu Arg Ala Leu Leu Ser Gln Leu Thr Lys lie Asp Gly Ser Leu Gly 285 290 295 cct aaa gaa tea cag agt tat gga gag ggt age tcg gag age ctc ctc 1024 Pro Lys Glu Ser Gln Ser Tyr Gly Glu Gly Ser Ser Glu Ser Leu Leu 300 305 310 acc gac atc ggt att cea GTT TGG AAT age TGC act tgatgatcga 1070 Thr Gly Asp He He Thr Val Ser Pro Trp Asn Cys 315,320 gtgtaacgag agttactatt gctatattcc tatccatgat tggaac att cttcgggggg 1130 aaataacgtg tgcttgtctg attgtacaaa catttcctca ctcttgtacc cacggtagat 1190 tcatgtaaaa taccaettat gacgctagac atacatatat ttcatcgtag ttccatttgt 1250 aaaaaaaaaa ttcaaaaaaa aaaaaaaggg cggccgc 1287 < 210 > 2 < 211 > 324 < 212 > PRT < 213 > Arabidopsis thaliana < 400 > 2 Met Glu Thr Glu Glu Glu Met Lys Glu Ser Ser He Ser Met Val Glu January 5 10 15 Ala Lys Leu Pro Pro Gly Phe Arg Phe His Pro Lys Asp Asp Glu Leu 20 25 30 Val Cys Asp Tyr Leu Met Arg Arg Ser Leu His Asn Asn His Arg Pro 35 40 45 Pro Leu Val Leu He Gln Val Asp Leu Asn Lys Cys Glu Pro Trp Asp 50 55 60 He Pro Lys Met Ala Cys Val Gly Gly Lys Asp Trp Tyr Phe Tyr Ser 65 70 75 80 Gln Arg Asp Arg Lys Tyr Ala Thr Gly Leu Arg Thr Asn Arg Ala Thr 85 90 95 Ala Thr Gly Tyr Trp Lys Ala Thr Gly Lys Asp Arg Thr He Leu Arg 100 105 110 Lys Gly Lys Leu Val Gly Met Arg Lys Thr Leu Val Phe Tyr Gln Gly 115 120 125 Arg Ala Pro Arg Gly Arg Lys Thr Asp Trp Val Met His Glu Phe Arg 130 135 140 Leu Gln Gly Ser His His Pro Pro Asn His Ser Leu Ser Ser Pro Lys 145 150 155 160 Glu Asp Trp Val Leu Cys Arg Val Phe His Lys Asn Thr Glu Gly Val 165 170 175 He Cys Arg Asp Asn Met Gly Ser Cys Phe Asp Glu Thr Wing Ser Wing 180 185 190 Ser Leu Pro Pro Leu Met Asp Pro Tyr He Asn Phe Asp Gln Glu Pro 195 200 205 Ser Ser Tyr Leu Ser Asp Asp His His Tyr He He Asn Glu His Val 210 215 220 Pro Cys Phe Ser Asn Leu Ser Gln Asn Gln Thr Leu Asn Ser Asn Leu 225 230 235 240 Thr Asn Ser Val Ser Glu Leu Lys He Pro Cys Lys Asn Pro Asn Pro 245 250 255 Leu Phe Thr Gly Gly Be Wing Be Wing Thr Leu Thr Gly Leu Asp Ser 260 265 270 Phe Cys Ser Ser Asp Gln Met Val Leu Arg Ala Leu Leu Ser Gln Leu 275 280 285 Thr Lys He Asp Gly Ser Leu Gly Pro Lys Glu Ser Gln Ser Tyr Gly 290 295 300 Glu Gly Be Ser Glu Be Leu Leu Thr Asp He Gly He Pro Ser Thr 305 310 315 320 Val Trp Asn Cys

Claims (19)

NOVELTY OF THE INVENTION CLAIMS

1. - An isolated nucleic acid comprising bases 89-1060 of SEQ DID NO: 1.

2. An isolated protein consisting of the amino acid sequence shown in SEQ ID NO: 2.

3. An isolated protein at least 70% homologous to said protein according to claim 2, further characterized in that said isolated protein is a functional counterpart of NACÍ.

4. A nucleic acid encoding the protein according to claim 3.

5. A transgenic plant which is transgenic for a nucleic acid comprising the nucleic acid according to claim 4.

6.- A plant cell transgenic which is transgenic for a nucleic acid comprising the nucleic acid according to claim 4.

7. A method of growing a plant comprising transforming said plant with the nucleic acid according to claim 4, and growing to said plant wherein said plant will grow larger than a wild-type plant.

8. - The method according to claim 7, further characterized in that said nucleic acid is controlled by a promoter activated by glucocorticoid.

9. The method according to claim 8, further characterized in that said plant is treated with glucocorticoid.

10. The method according to claim 9, further characterized in that the glucocorticoid is dexamethasone.

11. A method for growing a genetically altered plant larger than a wild-type version of said plant comprising the overexpression of NACI in said altered plant.

12. The method according to claim 11, further characterized in that said plant produces leaves larger than the version of said wild type.

13. The method according to claim 11, further characterized in that said plant produces roots larger than the version of said wild type.

14. The method according to claim 11, further characterized in that said plant produces more lateral roots than said version of said wild type.

15. A method for growing a genetically altered plant larger than a wild type version of said plant comprising the overexpression of a protein according to claim 3 in said altered plant.

16. - The method according to claim 15, further characterized in that said plant produces larger leaves than said wild-type version.

17. The method according to claim 15, further characterized in that said plant produces roots larger than said wild-type version.

18. The method according to claim 15, further characterized in that said plant produces more lateral roots than said wild-type version.

19. A transgenic plant made by the insertion of nucleic acid according to claim 4 within a wild-type plant.