Research area: Signal Transduction and Hormone Action

The theory that bioactive gibberellins (GAs) act as " inhibitors of inhibitors " of plant growth was based originally on the slender pea mutant (genotype la cry-s), but the molecular nature of this mutant has remained obscure. Here we show that the genes LA and CRY encode DELLA proteins, previously characterised in other species (Arabidopsis and grasses) as " repressors " of growth, which are destabilised by GAs. Mutations la and cry-s encode non-functional proteins, accounting for the fact that la cry-s plants are extremely elongated, or slender. We use the la and cry-s mutations to show that in roots, DELLA proteins effectively promote GA synthesis gene expression, as well as inhibit elongation. We show also that one of the DELLA-regulated genes is a new member of the pea GA 3-oxidase family, and that this gene appears to play a major role in pea roots.


INTRODUCTION
It is well known that primary root growth is strongly influenced by the plant hormone gibberellin (GA; Davies 2004). For example, the application of bioactive GA to roots treated with the growth inhibitor ancymidol completely restored growth to that of the untreated plants (Tanimoto, 1991). Yaxley et al. (2001) established the importance of GAs for root growth in peas by using a variety of GA-deficient mutant plants. In the na-1 mutant, for example, root GA 1 levels, and root elongation, were significantly reduced compared with WT plants, and when the GA 1 content was restored to WT levels, so too was root elongation.
Interestingly, there is also evidence that DELLA proteins promote the biosynthesis of active GAs. For example, in the Arabidopsis DELLA mutant rga, the expression of the biosynthesis gene GA4 is reduced, indicating that high DELLA protein levels are associated with an up-regulation of GA synthesis genes (Silverstone et al., 2001).
More recently Zentella et al. (2007) provided evidence that the Arabidopsis GA synthesis genes GA3ox1 (GA4) and GA20ox2 are direct DELLA targets.
DELLA proteins display conserved amino acid sequences among both dicot (Arabidopsis) and monocot (rice, wheat and barley) species (Silverstone et al., 1998;Gubler et al., 2002). However, the available evidence indicates greater redundancy in dicots compared with monocots (Ikeda et al., 2001;Thomas and Hedden, 2006). There have been five DELLA genes isolated from Arabidopsis (GAI, RGA, RGL1, RGL2 and RGL3), yet only one in wheat (RHT), rice (SLR1), barley (SLN1), and maize (D8) (Peng et al., 1997;Silverstone et al., 1998;Peng et al., 1999;Ikeda et al., In the current investigation we identify DELLA-encoding genes and their associated mutants from pea. We then use those mutants to show that in roots DELLA proteins promote the expression of GA synthesis genes and inhibit the expression of GA deactivation genes. We also report on the discovery of a previously unidentified GA 3-oxidase gene ( Fig. 1) from pea.

LA and CRY encode DELLA proteins
Although there has been much work on the involvement of DELLA proteins in shoots, no work has demonstrated the importance of DELLAs in roots. We sought to clone pea DELLA genes in order to study their involvement with GAs in the regulation of root growth. Partial sequences from PCR (see Materials and Methods) were used to probe a pea-shoot cDNA library. Full-length clones, selected on the basis of a conserved amino-terminus, were obtained in both cases, and sequence analysis showed that they both encode DELLA-like proteins. Sequencing of PCR products from genomic DNA from mutant genotypes showed that the base sequence of one of the two clones was altered in cry-s and cry-c plants, while the other was altered in la plants (Fig. 2). The mutant sequence altered in la plants co-segregated with the slender phenotype in a progeny in which LA and la segregated on a cry-s background (Supplemental Fig. S1). On this background, la segregates were immediately recognisable as slender plants while plants carrying at least one LA allele were WT in appearance. These data suggest that the first of the two cloned DELLA genes is LA.
In a separate cross segregating for both la and cry-s, each of the 7 slender (la cry-s) F 2 plants carried the mutant form of the second DELLA gene, as well as the for linkage between WA and CRY of 2.0 ± 1.1 (maximum likelihood method;Lamm, 1947). In a third cross, there was 100% co-segregation of wa and the second sequence (33 plants genotyped).
Furthermore, in accordance with other known DELLA proteins, LA and CRY both contain the DELLA motif, the TVHYNP motif, LZ, the VHIID domain and the LXXLL motif ( Fig. 2; Ikeda et al., 2001;Itoh et al., 2002).
The nature of the la and cry-s mutations was established by comparing the LA and CRY DNA sequences with those of the la and cry-s mutants. The la mutant was found to result from a 190-bp insertion at position Gln 85 (Fig. 2), and the cry-s mutation involves a frameshift deletion at position 152 ( Fig. 2). Both of these mutations are therefore predicted to encode non-functional proteins as a result of the out-of-frame stop codons. Another mutation in CRY, cry-c, involves a G to A substitution at base 583, which results in a glycine to glutamine substitution in the predicted protein. This in turn results in a reduced (but not abolished) capacity to inhibit growth: la cry-c plants are shorter than la cry-s plants (Reid et al., 1983).
As in the case of other multi-gene families, it would be expected that the gene pair, LA and CRY, arose from gene duplication. In fact, this gene pair is one of the first described examples of duplicate genes (Rasmusson, 1927). Interestingly, however, the duplication event appears to be quite ancient, having occurred prior to To investigate the roles of LA and CRY on root development, we recombined the severely GA-deficient mutant na-1 with la and/or cry-s. The na-1 mutant has been crucial for establishing a role for GAs in root development (Yaxley et al., 2001). This mutant has an extremely short shoot phenotype, termed nana, and roots that are shorter, thicker, and less ramified than those of WT plants (Yaxley et al., 2001). We found (Fig. 4) that recombining na-1 with la and cry-s essentially rescues the root phenotype of na-1. Interestingly, the la mutation on its own is able to largely rescue the na-1 root phenotype (Fig. 4, Supplemental Table S1), while cry-s on its own does not. This suggests that LA is the main functioning DELLA protein in the roots of pea.
The la mutation is also more effective at rescuing the shoot phenotype of na-1 plants, compared with cry-s (Fig. 4). It was previously noted that gene LA is a more effective inhibitor of shoot elongation than is CRY (de Hann, 1927;Reid et al., 1983). This difference is even more pronounced in the shoots of na-1 plants, and is especially clear in na-1 roots (Fig. 4).
In contrast to the roots, both of the null mutations la and cry-s are required to fully rescue the shoot phenotype of na-1 plants, and the shoot phenotype of na-1 la cry-s is slender (Fig. 4;Potts et al., 1985). The GA-saturated shoot phenotype of la cry-s plants confirms that LA and CRY are the only DELLA genes operative in the shoot. Results from genomic Southern blots probed with PsLA and PsCRY also did not provide evidence for more than two DELLA genes in pea. Here we found that na-1 la plants heterozygous for CRY/cry-s had internodes much longer than nana plants, and fitting within the range of Mendel's le-1 dwarf: thus la partially rescues the shoot phenotype of na-1. The dwarf phenotype of na la CRY/cry-s plants was not apparent in the previous investigation (Potts et al., 1985) because le-1 was also segregating. In contrast, we found homozygous na-1 la CRY plants had internodes only around half as long as the CRY/cry-s heterozygotes and a phenotype consistent with the upper end of the nana phenotypic range.
A novel GA 3-oxidase gene, PsGA3ox2, is expressed primarily in the roots and is responsible for the conversion of GA 20 to GA 1 Before investigating the effects of DELLA proteins on GA synthesis gene expression, we sought to clone additional 3-oxidase genes from pea. The reasoning for this was that the roots of the GA biosynthesis mutants le-1 and le-2 (null) are phenotypically similar to WT and contain similar levels of endogenous GA 20 and GA 1 to WT, in contrast to their dwarf shoot phenotype (Yaxley et al., 2001). It was therefore expected that another GA 3-oxidase must carry out substantial 3-oxidation in the roots (Yaxley et al., 2001). A previously unidentified pea GA 3-oxidase gene, PsGA3ox2 (Supplemental Fig. S2), was isolated using PCR primers based on Medicago sequence, 3' RACE and cDNA clones. The expression product of PsGA3ox2 converted [ 14 C]GA 20 to [ 14 C]GA 1 , as shown by HPLC and GC-MS-SIM, demonstrating its 3-oxidase activity (Supplemental Fig. S3).
The expression levels of the 3-oxidase genes PsGA3ox1 (also known as Mendel's LE; Lester et al., 1997;Martin et al., 1997) and PsGA3ox2 were measured in the shoot and root tissue of 6-day-old pea seedlings using real-time PCR.
PsGA3ox1 was more highly expressed in the shoot compared with root tissue, with an approximate 2-fold difference (P < 0.001, Fig. 5). Conversely, PsGA3ox2 showed approximately 2-fold higher expression in the root tissue than the shoot tissue (P < 0.02, Fig. 5).

Pea DELLA proteins promote the expression of GA synthesis genes and inhibit that of GA deactivation genes
In Arabidopsis shoots, DELLA proteins feed-back regulate the GA biosynthesis genes AtGA3ox1 and AtGA20ox1 (Dill and Sun, 2001;King et al., 2001;Silverstone et al., 2001). To investigate whether the pea DELLA proteins are involved in the feed-back regulation of key GA biosynthesis genes in pea roots, we undertook real-time PCR on LA and the mutant la on a cry-s background. In the la mutant, there was a 4-fold and 6-fold down-regulation of PsGA3ox1 (P < 0.001, Fig. 6A) and PsGA3ox2 (P < 0.05, Fig. 6A), respectively. The greatest effect on gene expression was seen for PsGA20ox1, which was down-regulated 14-fold in the mutant (P < 0.01, Fig. 6A). In contrast, a more than 2-fold up-regulation of the 2-oxidase genes PsGA2ox1 (P < 0.02, Fig. 6B) and PsGA2ox2 (P < 0.01, Fig. 6B) was seen in the mutant roots.
Similar results were obtained for CRY and the mutant cry-s on a la background (Supplemental Fig. S4).

DISCUSSION
DELLA proteins play a pivotal role in GA signal transduction (Fu and Harberd, 2003 mutations la and cry-s together produce the "slender" mutant, which played a pivotal role in the development of early theories on GA action. In 1957, Brian published the prescient theory that GAs, rather than positively promoting stem elongation, actually inhibit an inhibitor of that process. This was supported by genetic studies using GAdeficient mutants (Potts et al., 1985). Next came the discovery of growth-repressing DELLA proteins, and the finding that DELLAs are destabilised by GA (Harberd et al., 1998;Silverstone et al., 2001). Here we complete the picture by showing that LA and CRY encode DELLA proteins in pea. The mutant alleles la and cry-s both appear to encode non-functional proteins, and the stem elongation of la cry-s plants is similar to that of WT plants given a saturating dose of bioactive GA (Brian, 1957;Potts et al., 1985). It appears, therefore, that at least with respect to shoot elongation, LA and CRY are the only DELLA-encoding genes in pea.
It was shown previously that although the na-1 mutation dramatically reduces GA levels and leads to the very short "nana" phenotype, the la cry-s gene combination is completely epistatic to na-1 in shoots (Potts et al., 1985). Here we show that la crys also rescues (Fig. 4) the distinctive na-1 root phenotype (Yaxley et al., 2001).
Interestingly, la on its own largely rescues the na-1 root phenotype, at least in terms of elongation of lateral roots (Fig. 4). This indicates that LA may be the main functioning DELLA protein in roots.
We then used the pea DELLA mutations to examine the effects of these proteins on the expression of GA synthesis and deactivation genes in roots. In the DELLA slender mutants sln in barley (Chandler et al., 2002), slr1 in rice (Itoh et al., 2002), and la cry-s in pea (Martin, 1996), the synthesis of bioactive GAs is reduced, but this has only been shown for shoots. We therefore monitored the expression of GA biosynthesis and deactivation genes in the roots of WT and slender pea plants.
When both DELLA genes were null (la cry-s), there was a strong reduction in expression of the biosynthesis genes PsGA20ox1, PsGA3ox1 and PsGA3ox2, and an equally strong promotion of the deactivation genes PsGA2ox1 and PsGA2ox2, compared with LA cry-s or la CRY plants. Therefore, DELLA proteins promote the expression of GA synthesis genes and inhibit that of GA deactivation genes, indicating that in roots, DELLAs are an integral part of the feed-back and feedforward phenomena, whereby bioactive GA reduces GA synthesis and speeds up GA deactivation (Dill et al., 2001).
Another key GA gene from pea is Mendel's LE, also known as the 3-oxidase gene PsGA3ox1 (Lester et al., 1997;Martin et al., 1997). Mendel exploited the dwarf stature of mutant le-1 shoots in his original genetics experiments, but it is interesting to note that the roots of le-1 plants (and of other le mutants) are indistinguishable from the WT, and contain normal GA levels (Yaxley et al., 2001). The identification of a second GA 3-oxidase gene (PsGA3ox2) from pea provides an explanation for these observations. It appears that PsGA3ox2, which is relatively strongly expressed in roots, can compensate for the reduction in PsGA3ox1 activity in le-1 roots, and even for the complete loss of that activity in roots of the null mutant le-2 (Martin et al., 1997;Lester et al., 1999). The capacity for compensation by PsGA3ox2 (and possibly by other, as yet unknown, 3-oxidase genes) is clearly reduced in the shoots and consequently le-1 and le-2 shoots are dwarfed. However, it appears that there is some compensation in le-2 shoots, since they do produce a small amount of GA 1 .
In conclusion, we have isolated the LA and CRY genes of pea and have shown that they encode DELLA proteins. This provides valuable support for the "inhibitor of an inhibitor" model of GA action, which was based originally on the slender la cry-s mutant (Brian, 1957). Of the two DELLA genes, LA appears to be the major one operating in roots, as indicated by the capacity of la on its own to largely rescue the root phenotype of the GA-deficient na-1 mutant. We have used the la and cry-s mutations to show that in pea roots, DELLA proteins can be viewed as positive regulators of the expression of GA biosynthesis genes, including the newly discovered pea 3-oxidase gene, PsGA3ox2. Our studies on the pea DELLA proteins LA and CRY demonstrate the importance of GA signalling in the regulation of root growth.

Plant Material
Experiments involving gene expression studies and quantification of endogenous GAs were conducted with the tall (WT) Hobart line HL205+ (LA CRY; see . Progenies segregating for LA/la and/or CRY/cry-s were derived from the following crosses: HL133 (la cry-s NA) x NGB1766 (LA CRY na-1, Potts et al., from the same F 4 plant from cross HL133 x NGB1766. Other genotypes were selected from a cross between HL107 (LA CRY NA) and HL188 (la cry-s na-1; HL188 was selected from cross HL133 x NGB1766). The foundation lines HL2 (Lamm line 2), HL6 (Lamm line 6), HL7 (Lamm line 7) and HL8 (Lamm line 8a) were kindly provided in 1957 by Dr Robert Lamm, Alnarp, Sweden.

Plant Growth and Chemical Treatments
Plants to be raised to maturity for genetic studies were grown in a 1:1 mixture of dolerite chips and vermiculite, topped with pasteurized peat/sand potting mix. Plants for gene expression experiments were grown in 100% potting mix for 4-5 d. Gene expression material was immediately immersed in liquid nitrogen and stored in a -70ºC freezer.
PsCRY was obtained using nested PCR on cDNA, using primers based on the gene LS, a GRAS gene from tomato (DELLAs belong to the GRAS protein family).
The primary PCR was conducted with primers 5'-isolated by 3' RACE (Frohman et al., 1988). The partial sequence thereby obtained was used as a probe to screen approximately 350,000 clones of a pea seedling shoot library. A single clone containing the 5' end of the gene was isolated, sequenced, and then ligated into pGem-T Easy (Clonetech, USA) and expressed in E. coli. The functional activity of the expression product was tested as before (Lester et al., 1997), using [ 14 C]GA 20 as a substrate.

Segregation studies
The segregation of PsLA was followed using two PCR primers that flanked the deletion in the la mutant, 5'-CTTAGCTGTATTAGGTTATAAGGTTCGTT-3' and 5'-TCTTCACGAGTCTATCAGCAATCTT-3', giving a 542-bp band in WT, and a 727bp band in the la mutant. The segregation of PsCRY was followed using two PCR primers, 5'-CTTGAACAAGCTATGGGTAATTTTCA-3' and 5'-ATCCCTTTCTCCTGCGTT-3', which amplified a PCR product containing several Bcc I sites near the cry-s mutation, one of which was polymorphic between cry-s and the CRY gene in line 107 (Torsdag).

RNA extraction, cDNA synthesis and Quantitative RT-PCR
Plant material was ground to a fine powder with a mortar and pestle in liquid N 2 .
Approximately 100 mg of ground tissue was used for RNA extraction, as carried out by Wolbang et al. (2004). One to 2 µg of total RNA was used to synthesise singlestrand cDNA using the QuantiTect Reverse Transcription kit (Qiagen, USA). cDNA samples were diluted to a total volume of 100 µL.
For gene expression quantification, the following primer pairs were used:

Accession Numbers
Sequence data from this article can be found in the GenBank data libraries under accession number(s) DQ845340 (PsCRY), DQ848351 (PsLA) and DQ864759 (PsGA3ox2).

Supplemental Materials
Supplemental Figure S1. Co-segregation of the Slender Mutant and the LA/la Gene Pair.
Supplemental Figure S4. Effects of cry on Expression of GA Genes.
Supplemental Table S1. Length of lateral roots of pea genotypes.      Conserved domains and the mutation sites of la, cry-s, and cry-c are shown.