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. 2004 Sep;16(9):2463-80.
doi: 10.1105/tpc.104.022897. Epub 2004 Aug 19.

The SHINE Clade of AP2 Domain Transcription Factors Activates Wax Biosynthesis, Alters Cuticle Properties, and Confers Drought Tolerance When Overexpressed in Arabidopsis

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The SHINE Clade of AP2 Domain Transcription Factors Activates Wax Biosynthesis, Alters Cuticle Properties, and Confers Drought Tolerance When Overexpressed in Arabidopsis

Asaph Aharoni et al. Plant Cell. .
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Abstract

The interface between plants and the environment plays a dual role as a protective barrier as well as a medium for the exchange of gases, water, and nutrients. The primary aerial plant surfaces are covered by a cuticle, acting as the essential permeability barrier toward the atmosphere. It is a heterogeneous layer composed mainly of lipids, namely cutin and intracuticular wax with epicuticular waxes deposited on the surface. We identified an Arabidopsis thaliana activation tag gain-of-function mutant shine (shn) that displayed a brilliant, shiny green leaf surface with increased cuticular wax compared with the leaves of wild-type plants. The gene responsible for the phenotype encodes one member of a clade of three proteins of undisclosed function, belonging to the plant-specific family of AP2/EREBP transcription factors. Overexpression of all three SHN clade genes conferred a phenotype similar to that of the original shn mutant. Biochemically, such plants were altered in wax composition (very long fatty acid derivatives). Total cuticular wax levels were increased sixfold in shn compared with the wild type, mainly because of a ninefold increase in alkanes that comprised approximately half of the total waxes in the mutant. Chlorophyll leaching assays and fresh weight loss experiments indicated that overexpression of the SHN genes increased cuticle permeability, probably because of changes in its ultrastructure. Likewise, SHN gene overexpression altered leaf and petal epidermal cell structure, trichome number, and branching as well as the stomatal index. Interestingly, SHN overexpressors displayed significant drought tolerance and recovery, probably related to the reduced stomatal density. Expression analysis using promoter-beta-glucuronidase fusions of the SHN genes provides evidence for the role of the SHN clade in plant protective layers, such as those formed during abscission, dehiscence, wounding, tissue strengthening, and the cuticle. We propose that these diverse functions are mediated by regulating metabolism of lipid and/or cell wall components.

Figures

Figure 1.
Figure 1.
The shn Mutant and 35S:SHN1/WIN1 Plants Phenotype and Surface Permeability. (A) Mature rosette leaves of wild-type plants (ecotype Wassilewskija [Ws]) and the shn activation tag mutant on the left and right, respectively. (B) Chlorophyll extracted in 80% ethanol for 1 h from mature rosette leaves of shn progeny (left container) compared with wild-type leaves (right container). (C) Chlorophyll leaching assays with mature rosette leaves of shn and wild-type immersed in 80% ethanol for different time intervals. The results are derived from three independent experiments and depicted with standard error of the mean for each time point. fw, fresh weight. (D) Chlorophyll leaching assays as described above but using mature rosette leaves derived from 35S:SHN1/WIN1 (2-2) progeny and wild-type plants. (E) Rate of water loss from the progeny of the activation tag shn mutant, two 35S:SHN1/WIN1 primary transformants (2-2 and 2-5), and the wild type. Four rosette explants (root system and inflorescence stem detached) were weighed during the time intervals depicted. The results are derived from three independent experiments and depicted with standard error of the mean for each time point.
Figure 2.
Figure 2.
Changes in Wax Load and Surface Ornamentation of the shn Mutant Detected by Scanning Electron Microscopy. (A) and (B) Images of shn and wild-type mature adaxial side rosette leaves, respectively. Surface of shn is covered with regions of wax deposition, whereas wild-type surface is smooth and shows only little wax deposition. (C) and (D) Images of shn and wild-type cauline leaves after freeze fractionation, respectively. Cuticular ridges (indicated by arrows) could be detected on the surface of shn, whereas the wild-type surface is smooth. (E) and (F) Enlarged images of (C) and (D). Arrows point to the cuticular ridges in shn. (G) and (H) Images of silique surface derived from shn and wild-type plants, respectively. The surface of shn contains long ridges (indicated by an arrow) apart from wax crystals, whereas surface of the wild type is covered only by wax crystals. (I) and (J) Images of siliques after freeze fractionation derived from shn and the wild type, respectively. Cuticular ridges (depicted with arrows) could be detected on the surface of shn but not on the wild-type surface. (K) An image of the wild-type adaxial petal surface covered with conical cells containing cuticular ridge ornamentation. (L) An image of wild-type adaxial petal surface after freeze fractionation in which the cuticular ridges in the previous image (K) are detected (arrows). Compare these ridges to those detected by freeze fractionation of cauline leaves and siliques of shn ([C], [E], and [I]).
Figure 3.
Figure 3.
Chain Length Distribution (Percentage of Compound Class) for the Four Major Fractions in the Leaf Cuticular Wax of the Wild Type and shn. Level of significance obtained with a Student's t test is marked by the following: *, P < 0.1; **, P < 0.05; ***, P < 0.01.
Figure 4.
Figure 4.
Location of the Transposon Insertion in the shn Mutant and Activation of the Flanking Genes. (A) Location of the transposon insertion (inverted triangle) of shn on chromosome 1 between a gene with unknown function (At1g15350) and a member of the AP2/EREBP transcription factor family (At1g15360, SHN1). The distance of the enhancer (Enh) element in base pairs from the predicted ATG of the genes is also depicted. (B) RT-PCR experiments using oligonucleotides for amplification of the two genes flanking the transposon insertion in shn. Expression of both genes is strongly induced in rosette leaves of shn, whereas hardly any signal could be detected in two different wild-type plants. Amplification of the actin gene was used as a control for presence and levels of cDNA.
Figure 5.
Figure 5.
Recapitulation of the shn Mutant Phenotype and Detailed Morphological Changes in the 35S:SHN1/WIN1 Lines. (A) Rosette of wild-type and 35S:SHN1/WIN1 line at the same age (on the left and right, respectively). (B) Folded and twisted cauline leaves derived from a 35S:SHN1/WIN1 line. (C) and (D) A single flower of the wild type and 35S:SHN1/WIN1 line, respectively, display the folding of petals in 35S:SHN1/WIN1. (E) to (G) The adaxial surface observed using scanning electron microscopy of the distal part of a folded petal derived from a 35S:SHN1/WIN1 flower. The surface is composed of typical petal epidermis conical cells (marked with a square in [F]) in addition to unusually longer cells more than twice the size of a typical cell (marked with an ellipse in [F]). Unusual and typical conical petal cells are marked in (G). (H) The adaxial surface of a distal part of a wild-type petal (at the same magnification shown in [F]) with typical conical cells detected. (I) Seedling of a wild-type containing branched trichomes on the surface of its first and second pairs of true leaves. (J) Seedling of 35S:SHN1/WIN1 with hardly any trichomes on surface of first and second pairs of true leaves; the few detected are located on the blade margins and are single branched (marked with arrows).
Figure 6.
Figure 6.
The SHINE Clade of the Arabidopsis AP2/EREBP Transcription Factor Family. (A) Sequence alignment of the three Arabidopsis SHN proteins and their putative ortholog from rice (OsSHN1, accession number BAD15859). All four proteins contain a single AP2 domain at their N termini, a conserved middle domain (termed mm), and a conserved C-terminal domain (termed cm). Black background indicates 100% conservation, gray is 75%, and light gray is 50% conservation. (B) Phylogenetic analysis of the SHN clade protein members and other closely related AP2/EREBP family proteins from Arabidopsis, tomato (Lycopersicon esculentum) (LeERF1, accession number AAL75809), and rice (OsSHN1, accession number BAD15859). The scale bar of 0.1 is equal to 10% sequence divergence. Bootstrap values are given for nodes and are considered as a value for significance of the branches. Values higher than 850 are likely to be significant. (C) and (D) Plants overexpressing SHN2 and SHN3, respectively. Note the characteristic twisted appearance of the cauline leaves.
Figure 7.
Figure 7.
Expression Patterns of SHN1/WIN1 Detected in SHN1/WIN1 Promoter:GUS Lines. (A) An inflorescence. (B) Bottom half of a sepal. (C) Bottom half of a petal. (D) Bottom part of the stamen shown in (E). (E) A stamen. (F) Top part of the stamen shown in (E) and the anther and a pollen grain (inset). (G) Stage 16 silique. (H) Stage 17 silique; the nectaries are marked with an arrow. (I) An emerging lateral inflorescence.
Figure 8.
Figure 8.
Expression Patterns of SHN2 Detected in SHN2 Promoter:GUS Lines. (A) A dehiscing anther; dehiscence zone is stained blue. (B) to (D) Mature silique at stage 17; dehiscence zone is stained blue.
Figure 9.
Figure 9.
Expression Patterns of SHN3 Detected in SHN3 Promoter:GUS Lines. (A) An inflorescence. (B) Cauline leaf cut close to its proximal part with strong GUS staining at the cut edge. (C) Rosette leaf with cut and wounded edges either inside (made by punching a disk in the blade) or at both sides of the leaf do not stain for GUS. (D) Root of a 4-week-old plant grown in soil stained in the endodermis. (E) Lateral root of a 4-week-old plant grown in soil stained in the root cap. (F) Adaxial side of a young leaf derived from a 12-d-old seedling grown in vitro. Trichome support cells are stained blue.
Figure 10.
Figure 10.
Drought Tolerance Experiment with shn and 35S:SHN1/WIN1 Lines. Fifteen-day-old seedlings (six seeds sown per pot) of the wild type, progenies of shn, two 35S:SHN1/WIN1 lines (nos. 2-2 and 2-5), and a positive control rd29-DREB1A line (providing drought tolerance; Kasuga et al., 1999) were exposed for a period of 9 to 12 d of dehydration (DOD). Subsequently, seedlings were watered, and their appearance after a week (recovery) is presented in the image (apart from the first row at 9 d of dehydration, in which pictures were taken directly at the end of the dehydration period). The 9 d of dehydration results provide a clear difference between the wild type and shn as well as 35S:SHN1/WIN1, in which there is 100% recovery of the overexpression lines and 0% recovery of the wild type. Identical recovery results were obtained in an additional experiment comparing 50 seedlings of each wild-type and 35S:SHN1/WIN1 (no. 2-5) lines. See also Supplemental Table 1 online. n = 3 in the comparison between 35S:SHN1/WIN1 (no. 2-5) and n = 2 for all other comparisons.

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