2022
UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE BIOLOGIA VEGETAL
Patterns of suberization in root tissues of Arabidopsis thaliana mutants
Manuel Martins Cavaco
Mestrado em Biologia Molecular e Genética
Dissertação orientada por:
Professora Doutora Ana Milhinhos (orientadora externa)
Professora Doutora Célia Miguel (orientadora interna)
I Acknowledgments
I would like to thank the following people, without whom I would not have been able to complete this research. Forest Genomics and Molecular Genetics Lab team at Faculdade de Ciências da Universidade de Lisboa, especially to my supervisors Dr. Ana Milhinhos and Professor Célia Miguel, whose insight and knowledge into the subject matter steered me through this research (sorry for all the extra work Ana!). My lab colleagues, who have supported me and had to put up with my stresses and doubts. It truly has been very, very good time in this team.
Last but the most, my biggest thanks to my family for all the support you have shown me through this research and for the opportunity. To my girlfriend…you have been amazing, through all these years.
II Abstract
Suberin is a complex hydrophobic biopolymer found deposited in the bark of trees, which protects the tree by reducing water loss, regulating gas exchanges and from pathogens attacks. An extreme example of suberin deposition can be found in the bark (cork) of Quercus suber trees. In the root tissues,
suberization also occurs, protecting the root from biotic/abiotic stresses. There are mainly two suberized tissues in the Arabidopsis roots: the endodermis, which differentiates inwards to the cortex, close to the root apical meristem, and progresses along the root axis giving rise to another tissue, the periderm, a protective outer tissue more prominent at the root-hypocotyl border. Recently, a transcriptomic study revealed several promising candidate genes to be exclusively involved in cork differentiation in cork oak. However, the role of the candidates in the suberization process of tissues is yet to be
explored. By making use of the Arabidopsis simpler model system, the loss of function mutants for the for the homolog candidate genes was analyzed as o their endoderm and periderm development phenotypes. To investigate if the changes observed in the mutants were caused by alterations in suberin deposition patterns during primary growth at initial stages of endodermis development, the root patterns of suberization were analyzed by fluorescence microscopy using the fluorescent Fluorol Yellow 088 stain to seven-day old wild-type and mutant seedlings. Mutations in three of the candidate genes (WOX9/STIMPY, ANT and MYB36) led to alterations in suberin formation during developmental progression of the primary root endodermis. We found that especially WOX9/STIMPY, a homeobox gene required for shoot apical meristem growth, could be implicated in the differentiation of suberin in the endodermis. By examining the suberization patterns of the root endodermis of gain and loss of WOX9 function mutants’ results showed that while stip-1 and stip-2, the WOX9 loss-of-function alleles show delayed suberization of the endodermal cells, the gain-of-function mutant, stip-D, shows the opposite effect, when compared to the wild-type genetic background. Additionally, we performed expression analysis to genes encoding proteins of the suberin biosynthesis and deposition pathway in the WOX9 gain- and loss-of-function mutants, by quantitative real time-PCR to root-hypocotyl tissues. The gene expression data revealed that the anatomical defects observed in stip-1 are accompanied by the decrease in the steady-state mRNA transcript levels of FAR1, ASFT, CYP86A1, CYP86B1, KCS2, GPAT5, ABCG6 and GELP38 genes, hence, of all suberin-related genes that are representative of the suberin pathway, tested in this work. These results indicate that WOX9 might be operating in the suberin regulatory pathway well upstream of the biosynthetic pathway. Furthermore, to understand whether defects on endodermal suberization of the mutants were downstream of ABA we tested whether the exogenous application of ABA could rescue the stip-1 mutant phenotype. Indeed, 24h after ABA treatment, defects in stip-1 were partially overcome by ABA-induced suberization in the endodermal cells, suggesting WOX9 might operate upstream of ABA biosynthesis/signalling or bypass the pathway.
These results., together with preliminary phylogenetic analysis to WOX9, point towards a possible regulatory role for WOX9 gene during the suberization process, and we hypothesize on its positioning in existing regulatory pathways.
Keywords: Suberin, Periderm, STIMPY
III Resumo
A Suberina, que é um polímero hidrofóbico complexo encontra-se presente na casca de árvores, e desempenha diversas funções protetoras da planta, tendo papéis importantes na redução da perda de água por transpiração, na regulação de trocas gasosas e na proteção contra outros organismos potencialmente patogénicos. Um exemplo extremo da capacidade de deposição de suberina pode ser encontrado na casca do sobreiro (Quercus suber). Mas a suberina não é exclusiva dos troncos das árvores. Nas raízes de Arabidopsis thaliana também ocorre a deposição de suberina, conferindo à planta proteção contra stresses bióticos e abióticos. Nas raízes desta herbácea existem essencialmente dois tecidos suberizados: a endoderme, que se diferencia no centro do córtex, e que que se encontra perto do meristema apical radicular, cujo tecido se vai diferenciando ao longo do eixo da raiz até originar um outro tecido também suberizado, a periderme, que é o tecido protetor mais externo e que é mais proeminente na região de transição entre a raiz e o hipocótilo. Recentemente, um estudo de transcriptómica realizado no laboratório de acolhimento revelou vários genes candidatos promissores que poderão estar envolvidos na diferenciação da cortiça em sobreiro. No entanto, a função destes candidatos no processo de suberização ainda se mantém inexplorada. Usando a Arabidopsis como um modelo de estudo mais simples que o sobreiro, analisaram-se os mutantes de perda de função dos respetivos homólogos dos genes candidatos, mais especificamente, estudou-se o desenvolvimento da endoderme e da periderme. Para investigar se as alterações observadas nos mutantes eram causadas pelas alterações nos padrões de deposição da suberina durante o desenvolvimento primário, ou seja, durante as primeiras fases de desenvolvimento da endoderme, analisaram-se os padrões de suberização, na raiz, através de microscopia de fluorescência e usando um corante fluorescente (Fluorol Yellow 088), aplicado a plântulas com sete dias após germinação. Alguns trabalhos anteriores têm usado esta metodologia para estudar o envolvimento de genes candidatos no processo de suberização, estudando o impacto que possuem nas três zonas principais de suberização que estão presentes ao longo da raiz: em primeiro lugar, mais próximo do meristema apical da raiz existe uma zona não suberizada, onde não existem células da endoderme suberizadas; seguida de uma zona “patchy”, onde começa o aparecimento gradual de células suberizadas; e por último, uma zona contínua onde todas as células da endoderme se encontram totalmente suberizadas formando um anel contínuo que compõe a periderme. Analisando os perfis de suberização nos mutantes, observámos que as mutações em três genes candidatos (WOX9/STIMPY, ANT e MYB36) revelaram alterações na formação da suberina durante o desenvolvimento da endoderme, na raiz, relativamente aos controlos do tipo selvagem. Os restantes mutantes para os genes MYB84, ATHB6 e CLV1 não apresentaram diferenças significativas relativamente aos controlos, no entanto, não se sabe se podem influenciar a via da suberina alterando a composição química da mesma. Neste trabalho que apresentamos, foi evidenciado que o gene WOX9/STIMPY, que é essencial para o crescimento do meristema apical, poderia estar implicado na diferenciação da suberina na endoderme. Examinando os padrões de suberização nos mutantes de ganho e perda de função do WOX9, os resultados mostraram que enquanto os alelos de perda de função, stip1 e stip-2, mostram ter um atraso na suberização das células da endoderme relativamente aos controlos, o alelo de ganho de função, stip-D, manifesta o efeito contrário, quando comparado com o fundo genético do tipo selvagem e Col-0. A mutação de perda de função do gene ANT manifestou um fenótipo muito semelhante ao dos mutantes de perda de função WOX9, levando ao aumento da percentagem de células da endoderme na região patchy. O mutante de perda de função do factor de transcrição MYB36 manifestou um aumento na percentagem de células suberizadas, o que sugere que o MYB36 possa atuar como um regulador negativo do processo de suberização. Alguns trabalhos anteriores obtiveram padrões de deposição de suberina em mutantes myb36 fenótipos semelhantes aos obtidos neste trabalho. Tendo em conta estes resultados, foi feita uma análise de expressão, em genes que codificam enzimas
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envolvidas na síntese e deposição da suberina, em mutantes de ganho e perda de função do WOX9, através de PCR quantitativo em tempo real usando tecidos da raiz e do hipocótilo. Os dados referentes à expressão destes genes revelaram que os defeitos anatómicos observados anteriormente no stip-1 foram acompanhados pela descida dos níveis de transcritos dos genes FAR1, ASFT, CYP86A1, CYP86B1, KCS2, GPAT5, que estão envolvidos na biossíntese dos monómeros que compõe a suberina;
do gene ABCG6 e do GELP38, que estão envolvidos no transporte membranar e polimerização da lamela, respetivamente. Todos os genes representativos da via da suberina que foram testados nesta análise mostraram alterações relevantes relativamente à expressão nos controlos tipo selvagem. Estes resultados indicam que o WOX9 poderá operar na via regulatória da suberina a montante da via de biossíntese. O mutante de ganho de função, o stip-D não revelou diferenças significativas nos níveis de expressão da grande maioria dos genes da via da suberina, quando comparado com o controlo. Isto é indicativo que a atividade do WOX9 poderá ser reprimida por outros genes reguladores da via da suberina. Estudos prévios indicam que este gene poderá atuar reprimindo outros fatores de transcrição, pelo que seria interessante realizar, no futuro, um estudo de expressão semelhante, no entanto, com genes referentes aos principais fatores de transcrição envolvidos no processo de regulação da suberina.
Para explorar se estes defeitos observados nos mutantes estariam a jusante da via do ácido abscísico (ABA), testámos se a aplicação exógena de ABA reverteria o fenótipo observado no mutante de perda de função, o stip-1. Após 24 horas da aplicação exógena de ABA, os defeitos observados na suberização nos mutantes foram parcialmente recuperados através da indução da suberização mediada pelo ABA, sugerindo que o WOX9 poderá operar a montante da via de sinalização do ABA. Para explorar mais esta relação, deveria ser feito um estudo de expressão envolvendo os principais genes reguladores da via do ABA. Posteriormente foi feita uma análise filogenética por forma a explorar a história evolutiva destes genes candidatos e de forma e desmistificar qual poderá ter sido o seu envolvimento na colonização terrestre, por parte das plantas. Foram selecionadas nove espécies, previamente utilizadas em estudos referentes à origem evolutiva da suberina, de forma a representar três grandes grupos (Algas, Plantas-não-vasculares e Plantas Vasculares). A presente análise revelou a relevância de certos candidatos no processo da colonização terrestre. Tendo em conta os resultados obtidos nos ensaios anteriores, foi feito um aprofundamento na história evolutiva do gene WOX9 que revelou evidências de este estar envolvido em processos regulatórios nas primeiras plantas vasculares, porém a família deste gene revelou estar presente nas algas em fases mais precoces do processo de colonização terrestre, pelas plantas. Durante a análise histológica surgiram estruturas que eram indicativas de serem artefactos, no entanto o contrário foi posteriormente aprofundado. Em todos os genótipos, encontraram-se células únicas não suberizadas presentes em regiões completamente suberizadas e diferenciadas, parecendo tratar-se de um artefacto histológico, no entanto, trabalhos anteriores teriam já identificado estas estruturas como sendo células de passagem. Apesar de não se conhecer totalmente a sua função, admite- se a hipótese de estas células poderem ser pontos de interações bióticas e abióticas. Dada a ausência de suberização deduz-se que estas estruturas possam ser úteis, para no futuro construir uma linha marcadora repórter da expressão do WOX9 para explorar a possível função reguladora deste gene no processo de suberização, ou até mesmo de outros genes candidatos. O factor de transcrição WOX9 parece atuar numa via de sinalização complexa muito pouco explorada, onde as funções de muitos dos genes envolvidos não foram ainda esclarecidas. As hipóteses que foram colocadas parecem elucidar alguns aspetos desta via, no entanto, futuros trabalhos devem aprofundar as alterações observadas no presente estudo.
Palavras-chave: Suberina, Periderme, STIMPY
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Index
Acknowledgments ... I Abstract ... II Resumo ... III
1. Introduction ... 1
1.1 Suberin and its biosynthetic pathways ... 1
1.1.1 Aliphatic suberin pathway ... 1
1.1.2 Phenylpropanoid suberin pathway ... 2
1.1.3 Suberin-related monomers transport and layer formation ... 2
1.1.4 The regulatory network behind suberin biosynthesis and deposition ... 2
1.2.1 Meeting the candidates and their polymorphisms ... 4
2. Objectives ... 7
3. Materials and methods ... 8
3.1 Phylogenetic and evolutionary history analysis ... 8
3.1.1 Multiple sequence alignments and phylogenetic tree construction ... 8
3.1.2 Motif scan and family protein analysis ... 9
3.2.2 Whole-mount suberin histological staining ... 10
3.2.3 Fluorescence microscopy... 11
3.2.4 Image analysis ... 11
3.3 RNA extraction, purification, and cDNA synthesis... 11
3.4. Primer design and qRT-PCR optimization ... 11
3.5 Relative expression ... 12
3.7 Statistical analysis... 13
4. Results ... 13
4.1 Suberization patterns in loss of function mutants of the candidate genes ... 13
4.2 Suberin pathway genes expression in WOX9 mutant backgrounds ... 16
4.3 Evolutionary perspective on the candidate ... 17
5. Discussion ... 19
5.1 WOX9 affects the suberization pattern of the Arabidopsis roots ... 20
5.2 WOX9 affects suberization by altering expression of suberin biosynthesis and deposition- related genes ... 21
5.3 Old players, new tricks ... 22
5.4 Artefacts, or something more? ... 24 Index of images and tables ...………... VII
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6. Conclusion... 25 7. References ... 27 8. Supplementary information... 31
VII
Index of images and tables
Figure 1.1. Simplified Suberin Pathway Diagram in Arabidopsis. This scheme is based on previous literature and shows the two main suberin pathways and the involved genes in suberin biosynthesis reactions. The main transcriptional regulators of this process are represented inside the blue circle. In suberin lamellae, yellow strips correspond to the light lamellae composed aliphatic monomers and dark strips represent the dark lamellae that is made of phenolic compounds, predominantly, ferulates from the phenylpropanoid pathway. Dashed dark arrows represent more than a single enzymatic reaction.
Genes with question mark correspond to putative candidates to the respective process. Gene/Enzyme Abbreviations: KCS, β-KETOACYL-CoA SYNTHETASE; FAR, FATTY ACYL-CoA REDUCTASE;
CYP, CYTOCHROME P450; LAC, LONG-CHAIN ACYL-COA SYNTHETASES; GPAT, GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE; ASFT, ALIPHATIC SUBERIN FERULOYL TRANSFERASE; FACT, FATTY ALCOHOL: CAFFEOYL-CoA CAFFEOYL TRANSFERASE;
ABCG, ATP-BINDING CASSETTE FROM SUBFAMILY G and GELP, GDSL-TYPE ESTERASE/LIPASE PROTEIN. Adapted from Nomberg et al. (2022). ... 1 Figure 1.2. Suberin deposition pattern in the Arabidopsis primary root. (A) Seven-day old seedling stained with Fluorol Yellow and Aniline Blue counterstain under visible light at 10x magnification.
Scale bar, 1mm. (B) Same individual observed with fluorescence under UV light with a GFP filter. Non- suberized, patchy and continuous suberization zones are highlighted in white boxes. (C) Non-suberized zone, where the signal from FY is inexistent due to the absence of suberized structures. (D) Patchy zone is characterized by a chess-like pattern (E) continuous zone is a fully suberized portion of the root axis where all endoderm cells are fully suberized, as visualized by the strongest signal from FY. Scale bar correspond to 1 mm (A and B) and 100 µm (C, D and E) ... 3 Figure 4.1. Suberin deposition pattern analysis in A. thaliana roots of loss of function mutants.
Suberin was stained along the root axis, using FY in 7-day-old A. thaliana seedlings, and its quantification was inferred using three suberization zones: Continuous (brown), Patchy (grey) and non- suberized (black); (A) Arabidopsis seedling schematical representation showing suberization zones of the root. (B) Percentage of endodermal cells in each suberization zone / genotype. Mutants myb36-1, ant-9, stip-1, stip-2 and stip-D show differences in the process of suberization along the root axis. (D) Effect of exogenous ABA on stip-1 mutants, in the process of suberin deposition. 7 < N < 13; Error bars represent the standard deviation. The observed differences between zones in different genotypes were statistically validated using a conventional T-student test (p value < 0,0001 (****), p value < 0,001 (***), p value < 0,01 (**), p value < 0,05 (*)). Scale bars represent 1 mm. ... 14 Figure 4.2. Suberin biosynthetic gene expression is affected in WOX9/STIMPY mutants.
Quantitative Real Time PCR to selected genes involved in suberin biosynthesis and deposition. FAR1, ASFT, KCS2, CYP86A1, CYP86B1, ABCG6, GELP38 and GPAT5 mRNA transcript levels from root and hypocotyl tissues of two-month-old A. thaliana Col-0 and stip-1 and stip-D mutants are shown.
ACTIN2 was used as reference gene. Pfaffl method (Pfaffl, 2004) was used to calculate the relative gene expression. Significant differences were inferred using a regular Student T-test and are indicated at p value < 0,05(*) and p value < 0,01 (**). ... 16 Figure 4.3. Evolutionary perspective of candidate genes during plant land colonization. (A) The distinct protein domains present in the candidate genes, in A. thaliana, were searched in eight species and the number of domains found in each blastp top hit sequence was registered. Protein sequences from A. thaliana were used as query in NCBI blastp tool and all top hit sequences, obtained from the selected species, were analysed in InterProScan domain search tool. (B) The evolutionary history of WOX9 throughout plant land colonization was inferred through a Maximum Likelihood phylogenetic tree that include indicated species. The top hit sequences retrieved from the 8 species and additional paralogs obtained in Ensembl Plants database). were aligned using MUSCLE alignment tool. Maximum Likelihood method and Poisson correction model were used and the bootstrap consensus tree was inferred from 2000 replicates. Branches corresponding to partitions reproduced in less than 50%
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bootstrap replicates are collapsed. Scale bar indicates the number of substitutions per site and has a value of 1. This analysis was conducted in MEGA X version 11. Drosophila melanogaster is used as outgroup to root the tree. (C) Diagram showing different motif locations in protein sequences, from WOX9 phylogenetic tree, and respective levels of confidence. This analysis was conducted in Multiple Em Motif Elicitation software (MEME) in 10 protein sequences. Solid colour boxes represent motif sites predicted by MEME and used to build the motif. Only scanned sites with position p-values less than 0.0001 are shown. An additional table associating the motifs to their respective protein family was included and this analysis was performed using InterProScan. Only motifs that were related with a protein family are represented. Question marks were used to represent motifs where no domain family was associated. ... 18 Figure 5.1. WOX9 Evolution Throughout Land Colonization. (A) Diagram representing a proposed model by Lian et al. (2014) to explain the evolutionary history of WOX family. The schematic tree represents the different taxa that mark plant evolution and associate each one to three clades found in WOX protein family (Ancient, Intermediate and WUS). (B) Phylogenetic tree obtained in the present study, and proposed clades. It is possible to observe that the bryophyte group and the green algae group form a single cluster and the remaining angiosperms form another clade. Abbreviations: Bry: Bryophyte, Lyc: Lycopodiophyte, Gym: Gymnosperm, Mon: Monocots, Eud: Eudicots, Ppa: Physcomitrella patens, Smo: Selaginella moellendorffii, Pta: Pinus taeda, Osa: Oryza sativa, and Ath: Arabidopsis.
Image adapted from Lian et al. (2014). ... 24 Figure 5.2. Alterations in non-suberized-patchy transition zone patterns and passage cells. (A) The transition zone showing the “fade” like suberization pattern observed in most myb36-1 seedlings versus the wild-type pattern observed in Col-0. (B) Continuous zone of wild-type genotypes and the “passage cells”, that are highlighted with white arrows. In (A) and (B) the scale bar corresponds to 100 µm. ... 25
Table 1.1. Summary of candidate genes. TAIR accession and biological function are shown ... 5 Table 3.1. Selected species and sequences used in the construction of the phylogenetic tree. The taxonomy ID and sequence accessions were retrieved from NCBI database (www.ncbi.nlm.nih.gov/taxonomy). ... 9 Table 3.2. List of Arabidopsis gene specific primer list used for RT-qPCR analysis. Properties such as annealing temperature, extension time and primer amplification efficiency are included. ... 13
1 1. Introduction
1.1 Suberin and its biosynthetic pathways
Suberin is a lipophilic molecule crucial in plant development due to its unique properties that are product of its structure. The exact structure of suberin monomers was long uncertain and debated (Graça, 2015), but in the XXth century these doubts were cleared by gas chromatography and mass spectrometry in works of Kolattukudy and Holloway, pioneering the studies of suberin structure and composition (Kolattukudy et al., 1975; Holloway, 1983). Suberin is a chemically complex polymer not defined as having only one chemical structure. Briefly, this polymer is composed by two domains, a polyaliphatic and a polyaromatic, both being linked to each other and to a glycerol by ester bonds (Figure 1.1). These two groups are composed by various monomers which presence and composition vary among plant species (Graça et al., 2019).
Figure 1.1 Simplified Suberin Pathway Diagram in Arabidopsis. This scheme is based on previous literature and shows the two main suberin pathways and the involved genes in suberin biosynthesis reactions. The main transcriptional regulators of this process are represented inside the blue circle. In suberin lamellae, yellow strips correspond to the light lamellae composed aliphatic monomers and dark strips represent the dark lamellae that is made of phenolic compounds, predominantly, ferulates from the phenylpropanoid pathway. Dashed dark arrows represent more than a single enzymatic reaction. Genes with question mark correspond to putative candidates to the respective process. Gene/Enzyme Abbreviations: KCS, β-KETOACYL-CoA SYNTHETASE; FAR, FATTY ACYL-CoA REDUCTASE; CYP, CYTOCHROME P450; LAC, LONG-CHAIN ACYL-COA SYNTHETASES; GPAT, GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE; ASFT, ALIPHATIC SUBERIN FERULOYL TRANSFERASE; FACT, FATTY ALCOHOL: CAFFEOYL-CoA CAFFEOYL TRANSFERASE; ABCG, ATP- BINDING CASSETTE FROM SUBFAMILY G and GELP, GDSL-TYPE ESTERASE/LIPASE PROTEIN. Adapted from Nomberg et al., (2022).
The biosynthesis and deposition of suberin on plant cell walls, in form of lamellae, involves two different pathways that originate the two mentioned domains: the aliphatic pathway that originate aliphatic polyesters and the phenylpropanoid pathway from which aromatic polymers are formed (Figure 1.1).
1.1.1 Aliphatic suberin pathway
In the first via, suberin monomers are synthetized using long-chain fatty acids (LCFAS) precursors (C16:0, C18:0 and C18:1), this reaction is catalysed by the fatty acid synthase enzymatic complex, in -
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the plastids (Serra et al., 2022). The LCFAs are then acylated/converted to C16:0-CoA, C18:0-CoA and C18:1-CoA and exported to the endoplasmic reticulum (ER) and integrated into the suberin metabolism (Vishwanath et al., 2014). Part of the LCFA-CoA is elongated, by the Fatty Acid Elongase complex (FAE), originating very long-chain fatty acid (VLCFA). The 3-ketoacyl-CoA synthase gene family, where KCS2, KCS20 and KCS6 belongs, encodes an enzyme called ketoacyl-CoA synthase that is responsible, in the FAE complex, for originating VLCFA-CoA products of different chain lengths (Batsale et al., 2021). kcs2/daisy mutants show delayed growth and alterations in suberin composition (Li et al., 2007). VLCFA-CoA and the other part of LCFA-CoA are converted in suberin primary alcohols (>20:0C and 18:0C, respectively) by the activity of fatty acid reductases (FAR) such as FAR1, FAR4 or FAR5 (Domergue et al., 2010). The VLCFAs are further oxygenated by members of the CYTOCHROME P450 OXIDASE (CYP) family. In Arabidopsis thaliana cyp86a1/horst mutant roots the involvement CYP86A1/HORST in the biosynthesis of ω-hydroxyacids using LCFA-CoA as substrate has been shown (Höfer et al., 2008). CYP86B1/RALPH, a member of the same protein family, also demonstrated to be a key enzyme in the biosynthesis of ω-hydroxyacids and α, ω-dicarboxylic acids using VLCFA-CoA as substrate (Compagnon et al., 2009). Subsequently, glycerol, one of the major components of suberin, (Beisson et al., 2007), is attached to ω-hydroxyacids and α, ω-dicarboxylic acids in a reaction catalysed by GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE5 (GPAT5) that originate monoacylglycerol esters (Nomberg et al., 2022), one of the building blocks of suberin (Figure 1).
1.1.2 Phenylpropanoid suberin pathway
This via takes place in the cytoplasm (Figure 1.1) and its precursors are essential to originate suberin polyphenolic monomers. The general phenylpropanoid metabolism originate a diverse array of secondary metabolites (Vogt et al., 2010). A subset of these is composed by hydroxycinnamic acids such as coumaric, caffeic and ferulic acids, being this last one the predominant acids (Nomberg et al., 2022). ALIPHATIC SUBERIN FERULOYL TRANSFERASE (ASFT) is the enzyme, that catalyses the acyl transfer of feruloyl-CoA to x-hydroxy fatty acids and fatty alcohols (Molina et al., 2009 and Nomberg et al., 2022). According to previous works, FATTY ALCOHOL: CAFFEOYL-CoA CAFFEOYL TRANSFERASE (FACT), was demonstrated to use Caffeoyl-CoA as substrate to originate waxes (Kosma et al., 2012), particularly, n-alkyl caffeates.
1.1.3 Suberin-related monomers transport and layer formation
Although the mechanisms of synthesis of the suberin related monomers are well documented in Arabidopsis, the transport of these to the cell wall, where suberin lamella is formed, is not completely understood (Serra et al., 2022). Current literature suggests that the transport of these through the cytoplasmatic membrane into the apoplast environment is catalysed by an enzyme called ATP- BINDING CASSETTE TRANSPORTER (ABC), from the subfamily G. In Arabidopsis, ABCG2, ABCG6, ABCG11, ABCG16 and ABCG20 were already associated with the transport of suberin related monomers (Fedi et al., 2017) (Figure 1.1). The process of suberin polymerization in the cell wall is still very unclear (Nomberg et al., 2022), however, recent work from Ursache et al. (2021) in Arabidopsis, suggests the involvement of GDSL-TYPE ESTERASE/LIPASE proteins in this process, more specifically GELP38, GELP49, GELP51.
1.1.4 The regulatory network behind suberin biosynthesis and deposition
In the last decade, the family of MYB transcriptional factors has been shown to have an essential role in regulating suberin biosynthesis (Nomberg et al., 2022). In A. thaliana, the overexpression of MYB41 revealed an increment in suberin biosynthesis (Dylan et al., 2014). Later, a distinct subset of transcriptional factors composed by AtMYB107, AtMYB39 (SUBERMAN) and AtMYB9 were associated with the regulation of Arabidopsis roots endoderm suberization and seed coat (Cohen et al., 2020).
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Generating the quadruple loss of function mutant for MYB41, MYB53, MYB92 and MYB83 genes, resulted in the total absence of suberin in Arabidopsis roots, revealing the involvement of these transcriptional factors in the biosynthesis and deposition of this biopolymer. The family of MYB transcriptional factors is not the only one involved in the regulation of suberin synthesis, WRKY and ANAC protein families were also associated with this regulatory role (Krishnamurthy et al., 2020 and Mahmood et al., 2019). Many aspects of this complex regulatory network are still unclear, however, current literature suggests that one of the main strategies, to have a deeper insight about suberin biosynthesis and deposition regulation, is the genomic analysis accompanied by chemical structures analysis (Nomberg et al., 2022).
1.2 The periderm – The development of suberized tissues in Arabidopsis
During plant development, two main outermost tissues of the plant offer protection against abiotic and biotic stresses. During primary growth, the plant body is covered by the epidermis (protoderm), a protective tissue composed by a living and non-suberized parenchyma-made single cell layer.
Figure 1.2. Suberin deposition pattern in the Arabidopsis primary root. (A) Seven-day old seedling stained with Fluorol Yellow and Aniline Blue counterstain under visible light at 10x magnification. Scale bar, 1mm. (B) Same individual observed with fluorescence under UV light with a GFP filter. Non-suberized, patchy and continuous suberization zones are highlighted in white boxes. (C) Non-suberized zone, where the signal from FY is inexistent due to the absence of suberized structures. (D) Patchy zone is characterized by a chess-like pattern (E) continuous zone is a fully suberized portion of the root axis where all endoderm cells are fully suberized, as visualized by the strongest signal from FY. Scale bar correspond to 1 mm (A and B) and 100 µm (C, D and E)
Later in development, during secondary growth, the cambial activity and pericycle proliferation led to an increase in diameter of the root and this force causes the breakdown of the epidermis and its structural and protective function is assumed by a tissue composed by non-living and suberized cells, the periderm (Figure 1.2). The transition between these two outer tissues is accompanied by an increase in suberization along the root axis. During pericycle proliferation and xylem differentiation, endoderm undergoes a differentiation process due to compressing forces resulting from pericycle proliferation and an actively absorbing epithelium becomes, gradually, exclusively a protective tissue through a
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programed cell death, characterized by the formation and deposition of suberin (Barberon et al., 2016).
Afterwards, the gradual absence of endodermal cells and the pericycle activity lead to the formation of a continuous ring composed by non-living and suberized cells, the periderm. Previous work, involving suberin Fluorol Yellow (FY) histochemical staining of Arabidopsis roots has shown that the process of suberization creates a unique pattern along root axis when suberin is stained, allowing the individualization of three main zones of the root (Figure 1.2 B) The first zone is called “non-suberized”
and comprehends the portion between the root meristem and the first suberized endodermal cell (Figure 1.2 C). In this zone, the signal from the suberin marker is almost undetectable due to the inexistence of suberized structures. The second zone is named “Patchy” and this zone presents a peculiar chess-like pattern or, as described in previous works, “switch-like pattern” (Barberon et al., 2016). These patterns occur because the processes of suberization in the endodermis is gradual (Figure 1.2 D). The Patchy zone later develops into a third zone called “Continuous”, where all cells have suberin deposits in the form of suberin lamellae between the plasma membrane and the primary cell wall, creating a continuous suberized zone heavily signalized by suberin markers (Figure 1.2 E). The proportion of these three zones seems to be affected when suberin-related genes are disrupted (Shukla et al., 2021), causing the retardation or anticipation, according to gene´s function, of suberization in plant development. The analysis of proportion variation of these three main zones in loss of function mutants, could lead to new insights about their respective gene involvement in suberin biosynthesis and deposition.
1.2.1 Meeting the candidates and their polymorphisms
A recent transcriptomic analysis, using RNA-seq, to secondary growth tissues in Quercus suber, was performed with the objective of identifying specific candidate genes involved in cork cambium activity and phellem differentiation (Lopes et al., 2020). A high-resolution map of all transcripts was done and more than 6000 differentially expressed genes were identified. From this work, a short list of candidate genes (Table 1.1) was generated by analyzing the suberization in Arabidopsis secondary growth tissues (Susana Lopes, personal communication, unpublished). The selected candidate genes from the comparative transcriptomic analysis, were obtained investigating the cork oak (Q. suber) suberized cells.
However, functional analysis in cork oak is still quite challenging. As suberin related genes are common to all periderms from different organs or plant-species, such as those of Arabidopsis (Campilho et al.
2020), it has become increasingly common to use this model to study the suberization process (Ranathunge et al., 2011). This species is one of the most used models in plant research due to its short life cycle, easy cultivation, a short and a well described annotated genome, vast set of standardized procedures, such as for nucleic acids extractions, expression analysis and other laboratorial methods.
There is also high availability of genetic strains, marker lines and mutants and a huge diversity of databases and online tools/resources. Having these characteristics in consideration, A. thaliana is a great plant model that could be used to expand the molecular knowledge about suberin-related genes from Q.
suber, since many of these are common to both species. This would be especially relevant for Portuguese research, since cork oak is a species with a high market value due to its cork, has a high impact on the ecosystems and biodiversity (Bugalho et al., 2011) and is a species that have a high cultural and economic value to Portugal.
To assess the involvement of these genes in the suberization process, loss and gain of function mutants for the genes were examined as to their roots suberization patterns. ant-9 is a mutant of ANT that has an insertion in the second intron of the genic site (Randall et al., 2015), resulting in loss of function phenotype. Less vigorous growth and defective ovules are some of the characteristics of this mutant (Elliott et al., 1996).
5
Table 1.1 Summary of candidate genes. TAIR accession and biological function are shown.
Locus
TAIR
accession Function
Key References
AINTEGUMENTA
(ANT) Arabidopsis Essential in the development of gynoecium marginal tissues,
initiation/growth of ovules integumenta, development of female gametophyte and maintains
the meristematic competence of cells thus controlling the final size of each floral organ by controlling
their cell number.
Krizek et al., 2020;
Liu et al., 2000;
Krizek et al., 2021.
AT4G37750
HOMEOBOX PROTEIN 6
(ATHB6)
Arabidopsis Transcription activator that may act as growth regulators in response to drought stress.
Interacts with the core sequence 5'- CAATTATTA-3' of promoters in response to ABA. Involved in the negative regulation of the ABA
signalling pathway
Söderman et al., 1999;
Himmelbac h et al., 2002; Jiao et
al., 2022.
AT2G22430
CLAVATA1
(CLV1) Arabidopsis In association with CLV2 is involved in the detection of CLV3
and CLV3-like peptides, regulating shoot meristem maintenance and coordinating
growth between adjacent meristematic regions. Recently, CLV1 and CLV2 were associated
with Nitrogen stress response, during root development
Wang et al., 2020;
Hazak et al., 2016;
Bleckmann et al., 2010.
AT1G75820
MYB DOMAIN PROTEIN 36
(MYB36)
Arabidopsis Required for Casparian strip formation, subsequently regulating
global ion homeostasis, and transition from proliferation to differentiation state in the primary
root. SHORT-ROOT (SHR) together with transcriptional factor
MYB36 guide suberization, specifically, in the root endodermis of Arabidopsis.
Kamiya et al., 2015;
Liberman et al., 2016;
Wang et al., 2020.
AT5G57620
REGULATOR OF AXILLARY MERISTEMS3 (RAX3/MYB84)
Arabidopsis Key transcription factor involved in the regulation of axillary meristems formation, development
and may control lateral root development with their closest
homologs MYB37/RAX1 and MYB38/RAX2.
Guo et al., 2015;
Müller et al., 2006;
Wunderling
et al., 2018.
AT3G49690
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Table 1.1. (Cont.)
ANT is a transcription factor involved in floral development processes, upregulated by auxin response factors (Krizek et al., 2021). athb6 is a polymorphism of ATHB6, also a transcription factor that interacts with Abscisic Acid (ABA) signaling pathway, more precisely downstream of ABA INSENSITIVE 1, regulating an array of processes, such as such as seed dormancy and stomatal closure (Himmelbach et al., 2002). The loss of function mutant allele of CLAVATA1 used in this work was the clv1-1 that is characterized by a substitution of Gly 857 to Asp amino acid due to G to A substitution caused by the application of ethylmethane sulfonate (Clark et al., 1993). Individuals’ recessive for the mutations displays enlarged shoot apical meristem, increased numbers of floral organs and enlarged floral meristem (Williams et al., 1997, Clark et al., 2005 and Lindsay et al.,2006). This gene encodes a receptor involved in the detection of specific peptide signals like CLAVATA 3 and previous work suggests that CLV1 signaling pathway could be involved in root architecture regulation (Wang et al., 2020). myb36- 1 is a recessive mutant of MYB36 characterized by a putative rearrangement in the promoter region caused by fast neutron bombardment that led to changes to in leaf ionome and decreased expression of genes known to be involved in Casparian strip development (Kamiya et al., 2015). MYB36 is a master regulator that orchestrates endoderm differentiation, during root development, and is essential in Casparian strip formation (Kamiya et al., 2015). Previous work also indicates that MYB36 could interact with the auxin signaling pathway (Hormaeche et al., 2018). RAX3´s mutant allele rax3-1, is characterized by an insertion in the first exon that results in shoot branching defects in Arabidopsis (Müller et al., 2006). The RAX3 gene is expressed in response to drought and heat stress in cork oak (Wunderling et al., 2018), in the periderm of Arabidopsis roots (Wunderling et al., 2018) and is also involved in axillary bud formation during shoot development (Müller et al., 2006). In this work, three mutants of WOX9 gene were used: stip-1 is a loss of function mutant caused by the insertion into the first intron and recessive individuals for the mutation are characterized for having hyponastic cotyledons, reduced root development and smaller meristems (Skylar et al., 2010); stip-2 is another loss of function mutant and is characterized by a mis-sense mutation in codon 103 caused by the application of ethylmethane sulfonate and recessive individuals have a similar phenotype to stip-1, although it is less pronounced (Wu et al., 2005); and the only gain of function mutant used in this work was stip-D, that contains a pSKI015 construction that is inserted 270 bp downstream of transcription start site
(Wu et al.,2005) and mutants are characterized for having abnormal leaf shape, alterations in ovule and flower development andloss of apical dominance (Wu et al., 2005). WOX9 is a transcriptional factor involved in the regulation of meristematic stem cells. The lack of activity of this gene, in Arabidopsis, induce the exit of the cellcycle and promote premature differentiation, indicating, that WOX9 is crucial in tissue proliferation and pluripotency maintenance through cytokinin signaling (Wu et al., 2005; 2007).
WUSCHEL- RELATED HOMEOBOX 9 (WOX9/STIMPY)
Arabidopsis Transcription factor required for meristem growth and development. Also Promotes
cell proliferation during embryogenesis and prevents,
during postembryonic development, premature differentiation in meristematic
tissues. Involved in the transcriptional activation of
cytokinin response factors
Raines et al., 2016;
Wu et al., 2006;
Wu et al., 2007.
AT2G33880
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1.3 Suberin, one of the key players in plant land colonization
The process of land colonization by plants was certainly one of the most odd-defying stories in the course of life. Like all the great tales, many mysteries are yet to be solved, however, the success of this colonization is undeniable since c. 300 000 plant species form around 80% of the Earth´s Biomass (Christenhusz et al., 2016 and Bar-On et al., 2018). When plants started colonizing land, biotic and abiotic conditions were dramatically different, which raised many survival challenges such as osmotic control, climatic adaptation, biomechanical support, and protection against new and unknown organisms (Graça, 2015 and Pollard et al., 2008). A chemically complex and diverse molecule called suberin and its accumulation in cell wall of certain tissues, in form of lamella, certainly was crucial to mitigate these life-threatening challenges. The origin of this game changing polymer is still fairly unclear, however, one of the main strategies used to answer this question and understand the transition of plants from an aquatic environment to a terrestrial one is through studies of plant genomes and gene family structures in species that demonstrate suberin-related biosynthetic rudimentary systems (Philippe et al., 2020). One good example is found in Kondo et al., 2016 that demonstrated the presence of lipophilic layers composed by primitive wax lipids in a glycoprotein framework composed by acyl side chains in Klebsormidium nitens. These rudimentary suberin biosynthetic systems were also found in species that exist in semi-aquatic terrestrial environments, more specifically in the bryophyte group. In Physcomitrella patens the presence of CUS1, a protein involved in the biosynthesis of cutin, a polymer biochemically similar to suberin has been demonstrated (Philippe et al., 2020). In vitro, this protein, from P. patens, demonstrated cutin polymerizing capability through acyltransferase activity. These examples and many others helped demystify the origin and evolution of this biopolymer, however, many questions about this theme still unanswered. Current literature suggests that to solve all the challenges to a new and unknown environment an ancestral extracellular hydrophobic polymer was likely involved to form a cutin-like protective layer that posteriorly diverged to a new biopolymer denser, tighter and with a higher content in phenolic compounds, forming suberin-like defensive layers and providing crucial resistance to desiccation in the land environment (Philippe et al., 2020).
Exploring the evolutive history of each candidate gene could reveal eventual involvements of these in suberin rudimentary systems and in other processes that were crucial to land colonization a. In this work, histological and molecular approaches will be used explore the relationship between candidates and the suberin pathway, which together with the phylogenetic analyses can bring new insights into suberin regulation during development.
2. Objectives
The work hereby presented aimed to further contribute to elucidate the involvement of the selected candidate genes (Lopes et al., 2020) in the suberization process, by analyzing the respective homologs in A. thaliana to understand their putative involvement in suberin biosynthesis and deposition in the endodermis during early stages of root development. Further phylogenetic analysis was performed in different taxa to understand the participation of the most relevant candidate during land colonization.
The present work aimed to answer to the following questions:
• Are candidate genes RAX3, MYB36, CLV1, ANT, ATHB6, WOX9 involved in suberin deposition in the endodermis cells during root development? To answer this question, loss of function mutants were used in a screening to alterations in suberization patterns of endoderm in Arabidopsis roots.
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• Is WOX9 involved in the same regulatory pathway of suberin biosynthesis? To address this question a transcriptomic analysis of selected suberin related genes was performed in WOX9 loss and gain of function mutants in A. thaliana
• How might have the candidate genes contributed for the process of land colonization? To respond to this question a protein sequence analysis among different taxa was performed in order to understand the evolutionary history of candidates throughout land colonization.
3. Materials and methods
3.1 Phylogenetic and evolutionary history analysis
The selected species were gathered based on their presence in previous works about suberin evolution (Philippe et al., 2020; Niklas et al., 2017) and existence of the respective NCBI taxonomy ID. one Algae specie, two species of non-vascular plants and six Vascular Plants were collected. A total of nine species were gathered from three major groups (Table 3.1). The selection of the respective cork oak candidates’
homologs in Arabidopsis were obtained from unpublished data (personal communication from Susana Lopes). The following accessions were used in the present work, both in phylogenetic and histological analysis: ANT (AT4G37750), ATHB6 (AT2G22430), CLV1 (AT2G22430), MYB36 (AT5G57620), RAX3 (AT3G49690) and WOX9 (AT2G33880). ANT and CLV1 are Landsberg erecta (Ler) ecotypes and the remaining genotypes are in Colombia (Col-0) genetic background.
Each candidate genes’ protein sequence, in Arabidopsis, was retrieved from TAIR database (https://www.arabidopsis.org/) in FASTA format. Sequences were used to perform Protein BLAST (blastp) (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) in the nine above mentioned species.
The parameters used to select the sequences were based in previous works involving evolutionary analysis of different locus (Lian et al., 2003) and the E value of all the selected top hit sequences was bellow 1е-5. The sequences, of all species, where gathered from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/). A further search for paralogs was performed using the selected sequences of WOX9, obtained previously, as input in Ensembl Plants database (https://plants.ensembl.org/index.html).
The putative and translated protein sequences for the candidate genes, in Arabidopsis, and the top hit sequences for other species (Table 3.1) were scanned using InterProScan domain search tool (https://www.ebi.ac.uk/interpro/), an online tool that use predictive models provided by different databases to infer the presence of conserved domains in a specific input sequence.
3.1.1 Multiple sequence alignments and phylogenetic tree construction
To infer the evolutionary history of WOX9 and explore the relationship between the protein sequences in the different species, a phylogenetic analysis using a maximum likelihood tree was constructed (Goldman, 1990). All the protein sequences (Table 3.1), in FASTA format, were aligned using MUSCLE, a Multiple Sequence alignment online tool (https://www.ebi.ac.uk/Tools/msa/muscle/) (Edgar, 2004). The resultant alignment was uploaded to MEGA software, version 11 64 bits, (https://www.megasoftware.net/) and the main parameters for the tree construction defined. The statistical method used was the Maximum Likelihood, the phylogeny test was the Bootstrap method (Soltis et al.,2003)with a number of 2000 replicates, the substitution aminoacidic method used was the Poisson model and the heuristic method selected for this tree was the Nearest-Neighbor-Interchange.
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The resultant tree was exported in Newick format and uploaded in Figtree, version 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) (Rambaut, 2010) where the final tree was generated.
Table 3.1.Selected species and sequences used in the construction of the phylogenetic tree. The taxonomy ID and sequence accessions were retrieved from NCBI database (www.ncbi.nlm.nih.gov/taxonomy).
Species Name NCBI
Taxonomic ID Group Sequence Accession
Klebsormidium nitens 105231 Algae GAQ80365.1
Marchantia polymorpha 3197 Non-Vascular
Plant PTQ45519.1
Physcomitrella patens 3218 Non-Vascular
Plant BAM76366.1
Quercus Suber 58331 Vascular
Plant XP_023908458.1
Oryza sativa 4530 Vascular
Plant XP_025881188.1
Nicotiana tabacum 4097 Vascular
Plant XP_016462055.1
Brassica rapa 3711 Vascular
Plant XP_009143822
Amborella trichopoda 13333 Vascular
Plant
XP_020527832.1;
XP_020517625;
XP_006855139.1
Arabidopsis thaliana 3702 Vascular
Plant Q6X7J4.1
3.1.2 Motif scan and family protein analysis
In order to explore the biological and molecular role of these sequences, a motif scan was performed using MEME online research tool (https://meme-suite.org/meme/tools/meme). All protein sequences used to build the phylogenetic tree, were uploaded into MEME, and predicted motifs and corresponding p-values were calculated. To explore the biological role of the ten motifs, these were associated with specific protein families using InterProScan online tool. Motifs that could not be associated with any protein family were not included.
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3.2 Suberin in Arabidopsis genetic mutants for the candidate genes 3.2.1 Plant growth conditions
Arabidopsis seeds were first surface desinfected using a solution of commercial bleach (HClO) at 35% for 2 minutes, followed by 5 consecutive 2 min washes using sterile water. In a Laminar Flow Workstation (Microflow), 60-75 seeds were sown on 12 cm x 12cm Petri dish plates containing Murashige and Skoog basal salt medium (MS), supplemented with 1x MS Vitamins (Duchefa Biochemie), 0,8% (w/v) plant agar (Duchefa Biochemie), and 0.5% (w/v) crystalized Sucrose (Duchefa Biochemie) and adjusting the pH to 5.7. Particularly for wox-9 loss- and gain-of-function mutants (stip-1, stip-2, stip-D) seedlings were grown on similar MS medium with additional 15µg/L Phosphinotricin (PPT) (Duchefa Biochemie), to select the mutant seedlings with BASTA resistance. Plates were sealed with micropore tape (3M Micropore) and kept in obscurity, stratifying for 4 days at 4ºC to synchronize seed germination.
After that, seeds germinated and grew under a 16/8 h photoperiod (light/dark) for 7 days at 22ºC 60%
rH in a climatic chamber (Aralab) in an upright plate position. At least two plates were prepared for each growth experiment to eliminate plate positioning variation, and growth experiments were repeated twice in independent times. For each genotype, about 12-14 seedlings with proximate lengths were selected for further staining and analysis. For the Abscisic Acid (ABA) treatments, wox9 mutants were germinated and grown for three days on MS medium, supplemented with PPT and homozygous individuals selected, based on cotyledon curvature phenotype (loss of function mutants have smaller and hyponastic cotyledons (Wu et al., 2004)). At 0h, 12-14 seedlings were taken for histological analysis.
After one day, 12-14 seedlings were transferred to 1µM of ABA or mock treatment plates. After 24h of ABA/mock treatment, histological suberin staining (see section 3.2.2. for details) and observations under fluorescence microscopy analysis took place.
For the expression analyses, seven-day old seedlings from stip-1, stip-2 and stip-D were selected in MS medium supplemented with PPT and the corresponding wild-type control seedlings were transplanted to pots containing 3 parts peats: 1 part vermiculite (Projar) and grown for 2 months in a climatized growth walk-in chamber set to 60% RH and 16 hours of light and 8 of dark photoperiod. For each genotype, approximately 100 mg of root-hypocotyl tissue was harvested rom 24 individual adult plants. Six pools of four individuals’ tissues each were assembled as the biological replicates for the experiment. The pooled samples for all tissues were immediately frozen in liquid nitrogen and liquid nitrogen and stored at -80ºC until use.
3.2.2 Whole-mount suberin histological staining
To assess suberin deposition patterns in the root of the Arabidopsis mutants for the candidate genes, Fluorol yellow 088 (Brundrett et al., 1991; Lux et al., 2005) (FY, ChemCruz) (0.01% w/v, in lactic acid (LA) (Sigma-Aldrich) was used as a lipophilic marker (Barberon et al., 2016). The staining procedure was adapted from Barberon et al. (2016). Briefly, 10 mg of FY was dissolved per 100 mL LA, by heating to 70ºC in a lab oven (Thermo Scientific) during 10 min. Seedlings were submerged in the pre-heated solution at 70ºC during 30 minutes. Then, three consecutive 5 min washes in sterilized water to prevent the accumulation of FY granules in the root surface were performed. Aniline Blue (AB) (Duchefa Biochemie) was used as a counterstain by submerging the treated seedlings in 0.5% AB (w/v, in water) for 15 minutes. Four consecutive 7 min washes in sterile water removed the counterstain. All steps were performed in the dark. All the seedlings were placed in 0.25% Glycerol (Carl Roth) to preserve the tissues during slide preparation (slides from Thermo Scientific and cover slips from Carl Roth). Each seedling was picked from the glycerol and mounted on a slide in Antifade medium (1mL 10x Phosphate Buffered Saline (PBS) in 9 mL Glycerol (Merck KGaA) with 0,5 % (w/v) n-propyl gallate (Sigma- Aldrich) in dimethyl formamide (Sigma-Aldrich)), to delay photobleaching during the observation.
During all the procedure the seedlings were maintained in customized histological cassettes (HC) sealed
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with micropore (MP) to prevent seedlings from escaping during washes and stains. Suberin stain and anti-fade protocols used in this study are detailed in supplementary information sections 8.1 and 8.2.
3.2.3 Fluorescence microscopy
Olympus BX51 manual fluorescence microscope (Olympus) with a mercury fluorescence illuminator and Nomarski/DIC Prism for Transmitted Light was used to observe suberin deposits in the seedlings.
A GFP filter was used to detect Fluorol Yellow 088 fluorescence (excitation of 470-490 nm and emission of >515nm). All seedlings were observed at 10x magnification. A set of sequential photos was taken from each individual seedling using a TIS DFK 1.9MP CCD live color camera (Sony) and IC Capture software (https://www.theimagingsource.com/) (The Imaging Source), for posterior image stitching and analysis.
3.2.4 Image analysis
All sequential photos taken from each individual seedling were processed using Microsoft ICE (https://www.microsoft.com/en-us/research/project/image-composite-editor/). The software creates control points between images and aligns these, creating a unique image containing a single seedling.
The variation of suberization in each seedling was inferred delimitating 3 main zones (Non-suberized, Patchy and Continuous, as explained in the Introduction section). The length of each zone in each seedling was measured using ImageJ tool (imagej.nih.gov/ij). First, the images were set to the correct scale using the respective µm/pixel ratio from the Olympus BX51´s 10x objective, present in the FCUL microscopy facility (fculmf.campus.ciencias.ulisboa.pt/fculmf/widefield-fluorescence/olympus-bx51/).
Since roots are never mounted perfectly straight on slides, in order to minimize measure errors, each seedling was contoured by a selection tool and flattened using a straightening plugin. Then, the length of the 3 zones was registered for each individual biological replicate (seedling) for each genotype. For a better understating about this section, a video (https://youtu.be/RoNOfs3PRRQ)showing all the process and steps from image alignment to length measurement was produced.
3.3 RNA extraction, purification, and cDNA synthesis
About 65-100 mg of plant material from each tissue (leaves, base of the stem and root-hypocotyl in six pools of four individuals’ tissues), was ground to a fine powder, in liquid nitrogen, with the aid of pre- cooled mortar and pestles. RNA extraction was performed using the RNeasy Plant Mini Kit (Qiagen).
Briefly, to evaluate RNA quality and eventual DNA contaminations, total RNA was separated by electrophoresis on a 1% agarose/TAE gel. Then, extracted RNA was purified using the TURBO DNA- free™ Kit (Invitrogen) and the final yield of RNA for each pool was measured using Qubit RNA Broad Range Assay Kit (ThermoFisher) following the manufacturer´s protocol. To evaluate the efficiency of the TURBO DNA-free™ Kit and confirm RNA purity an additional electrophoresis was performed.
Complementary DNA (cDNA) was synthesized from 736 µg of total RNA using Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit #K1621 (ThermoFisher) with RevertAid M-MuLV Reverse Transcriptase (200 U/μL) (ThermoFisher) and Oligo (dT)18 primer (ThermoFisher).
3.4. Primer design and qRT-PCR optimization
Suberin biosynthesis pathway specific genes KCS2, GPAT5, ABCG6, CYP86A1, CYP86B1, ASFT, FAR1 and GELP38 were selected for expression analysis following Nomberg et al. (2022). ACTIN2 and GAPDH were used as housekeeping genes. Specific primers were designed for each transcript, using Primer3Plus (https://www.primer3plus.com/) and the properties of each pair of primers (melting temperature, self-annealing, Hairpin formation) were assessed using SMS PCR Primer Stat online tool (https://www.bioinformatics.org/sms2/pcr_primer/). The resulting set of primers used in this experiment are presented on Table 3.2.
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For each pair of primers, different extension times and annealing temperatures were tested to improve product amplification and primer specificity. The quality of the reaction was inferred visualizing the product in an Agarose Gel (2% TAE) and assessing band size and intensity. All the optimized qRT-PCR conditions for each gene can be found on Table 3.2. All optimizations were performed using a root cDNA pool from 3 samples diluted 1:10 (in DNase free water).
RT-qPCR was performed in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using optical 96-well BR White plates and SYBR Green Fluorophore (Bio-Rad). The reaction analysis was conducted in CFX Maestro software (Bio-Rad). The PCR reactions (10 µL total) included 8.5 µL 1X SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) plus 1.5 µL of the previously diluted cDNA. The following thermal-cycling protocol was followed for all PCR runs: initial denaturation step at 95°C for 3 min; 35 cycles of amplification: 95°C for 10s followed by 60ºC for 30s annealing/extension step (except for GAPDH and GELP38 amplification where 20 s were used instead). A dissociation step was performed after amplification to confirm the presence of a single amplicon (0.5ºC increment per 2-5 s, from 65ºC to 95ºC). Each PCR reaction included 3-6 biological replicates and 3 technical replicates each, controls (Positive control, Negative control and non-template control) to confirm absence of contaminations. For each primer pair, standard curves with serial dilutions (1/20, 1/50, 1/100, 1/200, 1/500, 1/1000, 1/10000) of template cDNA were obtained to determine the amplification efficiency (Eq. 3.1). SsoAdvanced Universal SYBR Green Supermix manufacturer recommend efficiencies between 90% and 110% for a robust RT-qPCR assay (Eq.3.2)
Eq.3.1 𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 (𝐸) = [10− 1𝑆𝑙𝑜𝑝𝑒] − 1
Eq.3.2
% 𝐸 = (𝐸 − 1) × 100%
3.5 Relative expression
Due to different amplification efficiencies for each set of primers, the Pfaffl method was used to calculate the relative expression of the genes tested (Pfaffl 2004). Briefly, to determine the expression ration between the sample (mutant) and the calibrator (wild-type) using one reference gene, the following equation was used:
Eq.3.3 𝑅𝑎𝑡𝑖𝑜 = (𝐸𝐺𝑂𝐼)∆𝐶𝑡,𝐺𝑂𝐼 (𝑐𝑎𝑙𝑖𝑏𝑟𝑎𝑡𝑜𝑟−𝑠𝑎𝑚𝑝𝑙𝑒)
(𝐸𝐻𝐾𝐺)∆𝐶𝑡,𝐻𝐾𝐺 (𝑐𝑎𝑙𝑖𝑏𝑟𝑎𝑡𝑜𝑟−𝑠𝑎𝑚𝑝𝑙𝑒) GOI – gene of interest HKG- housekeeping gene
The ratios mean and standard deviation observed among the biological replicates in each genotype were used to generate the graphical representations. Expression profiles for eight suberin-related genes (Table 3.2) were obtained in WOX9 mutants (stip-1 and stip-D) and corresponding wild-type roots (3 technical replicates for 3 biological replicates and an inter-plate correction factor, targeting the housekeeping gene ACTIN2).
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Table 3.2. List of Arabidopsis gene specific primer list used for RT-qPCR analysis. Properties such as annealing temperature, extension time and primer amplification efficiency are included.
Gene (Accession number)
Primer Annealing
Temp.
(Cº)
Extension Time (sec.)
Primer Efficiency (%)
GPAT5 (AT3G11430) Fw 5'-ACCGTGTCGCTAATTTGTTTGTTGG-3' Rv 5'-CCGTCGTGAAATATCACCGGAAGT-3' 60 30 109
FAR1(AT5G22500) Fw 5'-GGAGCCCTGAATGTTCTCAACTTCG-3' 60 30 104
Rv 5'-GAGAGTCTCCCCCATCTTGAATGGT-3'
KCS2(AT1G04220) Fw 5'-GCTTGAGAAAACCGGAGTGA-3' 60 30 101
Rv 5'-GAGAAGCATTGATCGGTCGT-3'
CYP86A1(AT5G58860) Fw 5'-TCGTTTACCTCAAGGCTGCT-3' 60 30 89
Rv 5'-GGAGTCTCAAACCGTTCACC-3'
CYP86B1(AT5G23190) Fw 5'-CCCGCTGATCACAAAGAGG-3' 60 30 101
Rv 5'-GAACCGTCCGTCTCTTAGCC-3'
ABCG6 (AT5G13580) Fw 5'-ATGAACCAACTTCGGGTCTG-3' 60 30 90
Rv 5'-CGGGACAAGAAGAGAAGACG-3'
ASFT (AT5G41040) Fw 5'-TACCAAACCCGATCCTGAAA-3' 60 30 90
Rv 5'-GCTCCAATTCCATCGAACAT-3'
GELP38 (AT1G74460) Fw 5'-ACGGGTTTGATAACTCGGATTCG-3' 60 30 101
Rv 5'-ACAATGTCGACGCTGGAATACACG-3'
ACTIN2 (AT3G18780) Fw 5'-GGCTCCTCTTAACCCAAAGG-3' Rv 5'-TTCTCGATGGAAGAGCTGGT-3' 60 30 106
3.7 Statistical analysis
For statistical analysis, GraphPad Prism software (version 9.0.0) (www.graphpad.com) was used. To infer significant differences between genotypes and root suberization zones, a non-parametric T-Student Test (p < 0.05, Mann-Whitney test) was performed. The same statistical test and software were used to infer the significant differences observed in the expression analysis performed.
4. Results
4.1 Suberization patterns in loss of function mutants of the candidate genes
To predict the involvement of the candidate genes found in Lopes et al. (2020), in suberin biosynthesis and deposition pathway, the patterns created by the three root suberization zones distribution (Non- suberized, Patchy and Continuously suberized) was analyzed in loss of function mutants for the candidate genes (Figure 4.1 A).
One of the candidates, CLV1, a leucine-rich receptor kinase that controls shoot and floral meristem size, and contributes to establish and maintain floral meristem identity, is known for playing a crucial role in regulating the expansion of the root system under N-stress conditions (Clark et al., 1997;
Williams et al., 1999; Araya et al., 2014). The distribution of each suberization zone in the clv1-1 loss of function mutant primary roots did not show major differences when compared with the Ler wild-
type genotype, in our growth conditions (Figure 4.1 B). Another candidate that has been shown to
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promote differentiation of the root endodermis is MYB DOMAIN PROTEIN 36 (MYB36), a transcriptional regulator shown to promote the development of the suberized Casparian band in Arabidopsis roots (Liberman et al., 2015). In the present screening, the myb36-1 loss of function mutant showed significant differences in the timing of suberization, with faster suberization occurring in myb36-1 mutant, revealed by an increased number of endodermal cells when compared to that of the control wild-type roots. Another candidate belonging to the class R2R3 MYB genes, the RAX3 transcription factor, has been shown to regulate axillary meristem formation (Müller et al., 2006) Although the rax3-1 mutant took longer to start differentiating the first suberized cells of the root, it simultaneously shows a faster entrance into fully suberized stage of endodermal cells when compared to the wild-type controls, as revealed by a shorter patchy region (Figure 4.1 B). Although this trend could be observed, none of these differences were statistically significant.
Figure 4.1Suberin deposition pattern analysis in A. thaliana roots of loss of function mutants. Suberin was stained along the root axis, using FY in 7-day-old A. thaliana seedlings, and its quantification was inferred using three suberization zones:
Continuous (brown), Patchy (grey) and non-suberized (black); (A) Arabidopsis seedling schematical representation showing suberization zones of the root. (B) Percentage of endodermal cells in each suberization zone / genotype. Mutants myb36-1, ant- 9, stip-1, stip-2 and stip-D show differences in the process of suberization along the root axis. (D) Effect of exogenous ABA on stip-1 mutants, in the process of suberin deposition. 7 < N < 13; Error bars represent the standard deviation. The observed differences between zones in different genotypes were statistically validated using a conventional T-student test (p value <
0,0001 (****), p value < 0,001 (***), p value < 0,01 (**), p value < 0,05 (*)). Scale bars represent 1 mm.
HOMEOBOX PROTEIN 6 (ATHB6) is a homeodomain leucine zipper class I protein that mediates ABA-response mechanisms, interacting with key components of signal transduction like ABI1, in Arabidopsis (Himmelbach et al., 2002). No significant differences were observed in the process of suberization along the root axis. The percentage of non-suberized cells was roughly the same (20%) in athb6 and wild-type roots. In the continuous zone of both genotypes a similar percentage of suberized
