Submitted by THIAGO MATEUS ROSA DOS SANTOS null (thiagomateusrp@gmail.com) on 2017-07-28T16:24:35Z No. of bitstreams: 1 Dissertação_Thiago_Mateus_Rosa_Santos.pdf: 2449824 bytes, checksum: 8934ae7010672e8bf2342056d81d2899 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-08-01T20:00:27Z (GMT) No. of bitstreams: 1 rosasantos_tm_me_jabo.pdf: 2449824 bytes, checksum: 8934ae7010672e8bf2342056d81d2899 (MD5) / Made available in DSpace on 2017-08-01T20:00:27Z (GMT). No. of bitstreams: 1 rosasantos_tm_me_jabo.pdf: 2449824 bytes, checksum: 8934ae7010672e8bf2342056d81d2899 (MD5) Previous issue date: 2017-05-03 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A cana-de-açúcar é acometida por uma doença conhecida por “escaldadura das folhas” causada pela bactéria colonizadora do xilema Xanthomonas albilineans, considerada uma das principais doenças que atingem a cultura da canade-açúcar. A sintomatologia na fase crônica se caracteriza, principalmente, pelo aparecimento de uma faixa branca ao lado da nervura central da folha, a qual evolui para clorose total causando a morte da planta. Uma vez que o patógeno pode ser transmitido de várias maneiras, o seu controle demanda altos custos. Desta maneira, o desenvolvimento de cultivares tolerantes é uma boa opção para o controle efetivo da doença. A tolerância e sensibilidade das plantas aos fatores bióticos está relacionada com a expressão de genes, e dentre estes, os miRNAs (incluindo o miR397 e o miR319) têm sido relatados como importantes reguladores em vários mecanismos de resposta das plantas. O objetivo deste trabalho foi analisar a expressão de dois miRNAs (miR319 e miR397) em duas cultivares de cana-de-açúcar (RB86-7515 - tolerante e SP78-4467 - suscetível), infectadas por uma linhagem de X. albilineans (IACXa11), considerada a mais virulenta do Brasil. Para isto, as plantas foram cultivadas em vasos, inoculadas com X. albilineans e mantidas em casa de vegetação. Amostras de folhas e colmos foram coletadas em cinco períodos (24, 72, 144, 360 e 720 h) e a expressão dos miRNAs foi analisada pela técnica de Stem-loop RT-qPCR. Os miR397 e miR319 apresentaram-se diferencialmente expressos nas cultivares e entre os tecidos. Na cultivar suscetível (SP78-4467), durante os primeiros períodos de infecção (24, 72 e 144 h), houve uma resposta tardia de defesa quando comparada com a cultivar tolerante (RB86-7515). O miR319 apresentou o mesmo perfil de expressão em folhas e colmos da cultivar RB86-7515 (tolerante), sugerindo que o reconhecimento do patógeno e a ativação dos mecanismos de defesa são modulados em ambos os tecidos. De maneira geral, as análises dos miRNAs demonstraram que a expressão do miR397 é menor quando comparada com o miR319. O mesmo padrão foi observado para os seus respectivos genes alvo. O miR397 regula a enzima lacase, importante na biossíntese de lignina. A repressão deste miRNA aumentaria a lignificação, sugerindo um mecanismo estrutural de resposta. O miR319 regula os fatores de transcrição (FTs) MYB e TCP, os quais são responsáveis pela sinalização de ácido abscísico (ABA) e ácido jasmônico (JA). A repressão destes hormônios vegetais desencadeia a sinalização por ácido salicílico (SA), o qual é responsável pela defesa contra patógenos hemibiotróficos, tal como X. albilineans. / Sugarcane is affected by a disease known as "leaf scald" caused by the bacterium Xanthomonas albilineans, which colonizes the xylem. This disease is one of the most important for sugarcane culture. The chronic phase is mainly characterized by the white band emergence along the central leaf vein, which causes total chlorosis of the leaf and plant death. Since the pathogen can be transmitted in many ways, his control demands high costs, and the development of tolerant cultivars is a good option for disease control. The plant tolerance and sensitivity to biotic factors is related to gene expression, and among these, the miRNAs (including miR397 and miR319), have been reported as important regulators in various plant response mechanisms. The aim of this work was to analyze the expression of two miRNAs (miR319 and miR397) in two sugarcane cultivars (RB86-7515 – tolerant, and SP78- 4467 - susceptible), infected by a strain of X. albilineans (IACXa11), the most virulent in Brazil. The plants were grown in vases, inoculated with X. albilineans and kept in a greenhouse. Samples of leaves and stems were collected in five periods (24, 72, 144, 360, and 720 h), and the miRNA expression was analyzed by Stem-loop RT-qPCR. The miR397 and miR319 expression were different between cultivars and tissues. In the susceptible cultivar (SP78-4467), during the first infection periods (24, 72 and 144 h), there was a late defense response when compared to the tolerant cultivar (RB86-7515). The miR319 presented the same expression profile in leaves and stems of the cultivar RB86-7515 (tolerant), suggesting that the pathogen recognition and defense mechanisms activation were modulated in both tissues. In general, miRNAs analyzes demonstrated that miR397 expression is lower when compared to miR319. The same pattern was observed for their respective target genes. The miR397 is a laccase regulator, important in lignin biosynthesis. Repression of this miRNA would increase lignification, suggesting a structural mechanism of response. The miR319 regulates the transcription factors (TFs) MYB and TCP, which are responsible for the abscisic acid (ABA) and jasmonic acid (JA) signaling. The repression of these plant hormones triggers salicylic acid (SA) signaling pathway, which is responsible for the defense against hemibiotrophic pathogens, such as X. albilineans. / CNPq: 153785/2014-4

Growth polarity in leaves – a final discussion Insights into the growth processes of leaf lamina have come from studies on several species including Arabidopsis, Antirrhinum, tobacco and maize. A feature common to the growth of leaf in these distantly related species is the existence of a pronounced growth gradient in the proximo-distal axis -growth at the tip (distal part) is arrested at an early stage while the basal region (proximal part) continues to grow for the longest duration. This is because the cell division is arrested first at the tip at an early stage of development and the arrest progressively spreads towards the base. Along with the strong proximo-distal growth gradient, a milder growth gradient also exists in the medio-lateral axis, such that the cell division arrest travels slightly faster on the leaf margins imparting an overall convex shape to the arrest front. The temporal and spatial progression of the arrest front has not only been implicated in shaping up of a leaf but is also of paramount importance in the maintenance of a flat surface during leaf growth. Although the patterning mechanisms described above seem to operate during leaf growth in many6 species, the molecular mechanisms governing these processes is still in its infancy. Moreover, patterning of leaf growth has been studied only in a handful of model species and, therefore, the information from the vast body of natural variation remains neglected. Proximo-distal growth patterning by CINCINNATA Mutant leaves with altered rates or shapes of the arrest front progression deviate significantly from the normal shape and overall flat structure. Mutation in the CIN gene in Antirrhinum and its orthologues in Arabidopsis cause buckling of the leaf due to excess cell proliferation, which in turn is caused by a delayed progression of the arrest front. CIN-like genes code for TCP transcription factors and are expressed in a broad zone of a growing leaf somewhat distal to the proliferation zone. Even though several direct and indirect targets of CIN-like genes have been identified in various plant species, their role in regulating leaf maturity and surface curvature has remained unclear. The comparison of global transcription profile of wild type and cincinnata mutant of Antirrhinum showed that the expression of genes involved in either signaling or biosynthesis of the major growth hormones were altered in the mutant. By combining DNA-protein interaction, expression analysis, chromatin immuno-precipitation and RNA in situ hybridization, we show that CIN maintains surface flatness by regulating the signaling or level of major plant hormones in developing leaves. CIN promotes cytokinin signaling by directly binding to and thereby promoting the expression of a cytokinin receptor, AmHK4, in a spatio¬temporal manner. Furthermore, it also seems to affect GA level indirectly in young leaves by regulating the spatio-temporal as well as levels of GA-biosynthetic and GA-degrading enzymes. Thus, CIN seems to accelerate maturity in leaf cells along the tip-to-base direction through its effect on the cytokinin and GA signaling pathways. In addition, CIN suppresses auxin signaling more at the margin than in the centre by establishing a margin-to-medial expression gradient of a homologue of the auxin suppressor IAA3, thereby suppressing excess cell proliferation on the margin. Our results uncover an underlying mechanism in a developing leaf that controls curvature of the leaf surface by promotion of timely exit from cell proliferation in the proximo-distal as well as the medio-lateral axes via multiple hormone pathways. Divergent growth polarities in the proximo-distal axis of leaves The morphogenetic gradient in the proximo-distal axis of a leaf is brought about by the dynamic expression of several heterochronic regulators which can include TCP and GRF classes of transcription factors. Interestingly, many of these transcription factors are also regulated post-transcriptionally by micro RNAs. In case of the studied model species, these factors seem to be associated with basipetal growth. The early arrest in cell proliferation at the tip and continued cell division at the base has served as a paradigm in studying leaf growth and has been used to conceptualize the growth of leaves with different shapes. However, the possibility of the existence of different patterning mechanisms during leaf growth in the highly diverse plant kingdom remains unexplored. Our survey of leaf growth patterns in 75 dicot species reveals the existence of four distinct proximo-distal polarities in growth patterns. Using the law of simple allometry, we also show that the differentially growing regions of leaves bear a constant relationship between them during growth. A combination of cell-size studies, histochemical staining and expression analysis reveals a strong correlation among growth pattern, cell size and the cell proliferation status. The cell size studies also indicate that there is a wide variation in the final cell sizes of leaves and the relative contribution of cell division and cell expansion to the final leaf size can be highly variable. Furthermore, we find that the varying growth patterns are linked to changes in the expression pattern of miR396, which controls the expression pattern of cell division regulatory transcription factors, the GRFs. Mis-expressing miR396 at the base of the young Arabidopsis leaf caused an early exit from cell division while reducing the expression of the miR396 at the tip allowed cell division to continue for a longer duration near the tip. Our results demonstrate that leaves with similar shapes can be differently patterned and that this divergent patterning is linked to the expression differences in the regulatory micro RNA, miR396 In conclusion, this study shows that regulators like CIN maintain surface flatness of the Antirrhinum leaf during growth by promoting timely exit from cell division along the proximo-distal and the medio-lateral axes; and it does so by regulating multiple hormone pathways. Although the basic mechanism of patterned cell division and differentiation seems to be conserved among species, the polarities of growth can vary. The variability in the growth polarities could be brought about by changes in the trans-regulation or cis-regulatory changes in the patterning genes.