Memoria para optar al título profesional de Ingeniero Agrónomo / Chile necesita variedades de arroz (Oryza sativa L.) que se adapten mejor a las condiciones de clima, suelo y manejo del país, como por ejemplo variedades de ciclo más corto y de alto rendimiento que las existentes. En este sentido, la empresa Maqsarroz introdujo en Chile una nueva variedad de arroz, Sandora, proveniente de Hungría. En esta memoria se estudió el efecto de la fecha de siembra, la dosis de nitrógeno y de semilla en las variables biomasa, rendimiento, peso 1.000 granos, granos por metro cuadrado, panículas por metro cuadrado, granos por panícula y porcentaje de granos llenos. Se utilizó un diseño estadístico de parcelas divididas en bloques completamente aleatorizados, la parcela principal fue la fecha de siembra, la sub-parcela la dosis de nitrógeno y la dosis de semilla ocupó la sub-sub-parcela. Los resultados obtenidos muestran que una combinación correcta de fecha de siembra y dosis de nitrógeno permite alcanzar los mayores rendimientos. Una siembra temprana fertilizada con 100 kg N ha-1 obtuvo el mayor rendimiento, 9.3 Mg ha-1, lo que contrasta con una siembra tardía que también fue fertilizada con 100 kg N ha-1, pero en la que se obtuvo un rendimiento de 6,2 Mg ha-1. En lo que respecta a la dosis de semilla, independiente de las dosis utilizadas, cuando se complementó con 100 kg N ha-1 en todos los casos se obtuvieron rendimientos entre 7,3 y 7,8 Mg ha-1.

Cadmium (Cd) is one of the major contaminants in agricultural soil, threatening agricultural production and human health. The objectives of this research work were to understand the tolerance mechanism in rice (Oryza sativa L.) genotype with more Cd-tolerance, and the relation between changes of peroxidases activities and peroxidase gene expression profiles after Cd-treated. For more, we analysis about cis-acting elements in the rice peroxidase genes promoter sequences region, gene structure of rice peroxidase genes and phylogenic relation among 9 peroxidase genes which were blasted from 6 Arabidopsis thaliana Cd-induced based on the peroxidase genes protein sequences. We used 9 upland and 32 varieties rice seeds as materials for germination and the growth of seedlings test with 50 or 500 £gM CdCl2 application, respectively. Rice seeds germination is a complex physiological and biochemical process, and is highly affected by cadmium. The results showed that Cd inhibited both the growth of radicles and coleoptiles. At germination stage, Cd highly inhibited the growth of upland rice. Among these rice varieties, Japonica type cultivars are more tolerant to Cd, but Indica type are more sensitive to Cd. Upland rice cultivars are the most sensitive to Cd. At seedling stage, Cd highly inhibited the growth of roots, but slightly inhibited the growth of shoots. To cope with Cd-induced stresses, plants adopt different strategies and posses a variety of defense mechanisms to prevent themselves from Cd damage. Peroxidase (POXs) is an important antioxidative enzyme for defense responses against Cd oxidative stress. The results suggested that different rice variety has a specific peroxidase gene expression pattern by the pI focusing electrophoresis after Cd-treated, and the peroxidase activities are significant differences when these rice varieties faced Cd stress. In this study, we searched the rice databases (japonica type) and cloned the promoter sequences of the indica type of the rice peroxidase genes, pI 4.5 POX and pI 5.1 POX genes. According to the search results about cis-acting elements from PLACE and PlantCARE databases, these cis-acting elements can be divided into three classes, showing as follow¡G1. Transcriptional related; 2. Light regulated related, and 3. Plant hormones- and stress-related. Based on all reported cis-acting elements, there are many types of transcription factors (TFs) involved in regulation of rice pI 4.5 POX and pI 5.1 POX genes expression. These TFs, which can recognize the specific cis-acting elements to regulate the gene expression, and will be induced by stresses and defense-related plant hormones. We found a cis-acting element (CURECORECR) which response to copper in both rice peroxidase genes promoter regions. There is a number of difference between Japonica type and Indica type peroxidase genes, japonica type peroxidase has more cis-acting elements than indica type. Plant class III peroxidases are present in all land plants. All land plant peroxidase genes are with the same putative ancestor of peroxidase genes and are orthologous genes, but they have specific functions of individual perxidase genes owing to their promoter sequences are very divergent. We used 6 Arabidopsis Cd-related peroxidases protein sequences as a starting point for rice peroxidase datas mining. We found 9 rice peroxidase genes have a closely relation among them. For more details about these rice peroxidase genes, searching each one of these rice peroxidases its gene structure on the Rice Genome Annotation Project (RGAP), comparison and their relation. The expressions of these peroxidase genes are very different among them after Cd treatment, and we also found the same cis-acting element (CURECORECR) which response to copper in all 9 rice peroxidase genes promoter regions. There is a number of difference among them.

Cd significantly inhibited the growth of both rice cultivars. The Tainung 67 cultivar is more tolerant to Cd than Taichung 1 cultivar after 48 h incubation in CdCl2 solution. The Cd tolerant cultivar¡XTainung 67¡¦s PODs in roots might synthesize more lignin in Cd-treatments. Meanwhile, the decrease of H2O2 levels is accompanied with the enhancement of POD activity in Cd-treated tissues. PODs here might also remove excess H2O2, thus serving detoxifying role and synthesizing more lignin for protection. In Taichung 1 cultivar, the accumulation of H2O2 in Cd-treated tissues could be due to the less amount of POD enhancement induced by Cd. In response to Cd treatment, the Taichung 1 cultivar also synthesizes little lignin, and therefore is Cd-sensitive.

Copper-treated rice seeding (Oryza sativa cv. Tainung 67 and Taichung native 1) showed significant inhibition in rice root growth, and an enhancement in POD activity. POD within Tainung 67 rice roots might synthesize more lignin in Cu-treated tissue. Meanwhile, the decrease of H2O2 levels is accompanied with the enhancement of POD activity in Cu-treated tissues. The increase in POD activity induced by Cu might remove excess hydrogen peroxide serving a detoxifying role and synthesizing more lignin for protection. In Taichung native 1 rice cultivar, high amounts of H2O2 accumulated in Cu-treated tissues could be due to the less amounts of POD induced by Cu. In response to Cu treatment, the Taichung native 1 cultivar also synthesizes a little lignin, and is more Cu-sensitive. Therefore, The Tainung 67 cultivar is more tolerant to Cu than Taichung native 1.

Cadmium-treated rice seeding (Oryza sativa L. cv. Taichung Native 1 and O. sativa L. cv. Tainung 67) showed inhibition in the growth of rice roots or leaves, and an enhancement in POX activity. In Tainung 67 cultivar, Cd treatment may have influence over cis-regulatory elements in POX promoter region and enhanced transcription of POX or enhance glycosylation of POX. The increase in POX activity induced by Cd might remove excess hydrogen peroxide serving a detoxifying role and synthesizing more lignin for protection. In Taichung Native 1 cultivar, high amounts of H2O2 accumulated in Cd-treated tissues could be due to the less amounts of POX induced by Cd. In response to Cd treatment, the Taichung Native 1 cultivar also synthesizes a little lignin, and is more Cd-sensitive. Therefore, the Tainung 67 cultivar is more tolerant to Cd than Taichung Native 1.

Aluminum-treated rice seeding (Oryza sativa L. cv. Taichung Native 1 and O. sativa L. cv. Tainung 67) showed significant inhibition in rice root growth, and an enhancement of POX activity. In Tainung 67 cultivar rice synthesizes more lignin in Al-treated tissue. Meanwhile, the decrease of H2O2 levels is accompanied with the enhancement of POX activity in Al-treated tissue. The increase in POX activity induced by Al might remove excess hydrogen peroxide serving a detoxifying role and synthesizing more lignin for protection. In Taichung native 1 cultivar, high amounts of H2O2 accumulated in Al-treated tissues could be due to the less amounts of POX induced by Al. In response to Al treatment, the Taichung native 1 cultivar synthesizes less lignin in comparison with that of Tainung 67, and is more Al-less tolerant. Therefore, the Tainung 67 cultivar is more tolerant to Al than Taichung native 1.

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O arroz é o alimento base para metade da população mundial. Entretanto, é uma fonte pobre em micronutrientes essenciais como Fe e Zn, que são removidos durante o processamento do grão para produção de alimento. Por essa razão, populações cujas dietas baseiam-se principalmente em arroz estão sujeitas à deficiência de Fe e Zn, que são as deficiências nutricionais mais comuns no mundo, afetando mais de dois bilhões de pessoas. Uma vez que as folhas-bandeira são uma das fontes de remobilização de metais para os grãos em desenvolvimento, a identificação dos mecanismos moleculares que contribuem no processo de transporte de metais das folhas-bandeira para os grãos pode ser útil em programas de biofortificação. Dessa forma, utilizamos duas abordagens diferentes para estudar esta questão. Primeiro, realizamos uma análise por hibridização subtrativa supressora (SSH) em folhas-bandeira de duas cultivares de arroz e isolamos 78 sequências induzidas no estágio de enchimento do grão (R5) em relação ao estágio de emergência da panícula (R3). A expressão diferencial de alguns genes selecionados (entre eles do fator de transcrição OsNAC5) foi confirmada por PCR quantitativo. Mostramos que a expressão de OsNAC5 é ativada em processos de senescência natural (envelhecimento) e induzida (escuro, aplicação de ABA, alta salinidade, frio e deficiência de Fe) e sua expressão não é afetada na presença de 6- benzilaminopurina (um inibidor de senescência) em condição de senescência induzida por escuro. A indução da expressão de OsNAC5 sob alta salinidade é abolida por nicotinamida, um inibidor dos efeitos do ABA. Este resultado e a presença de elementos cis-atuantes na região promotora do gene OsNAC5 sugere uma regulação dependente de ABA. Utilizando quatro diferentes cultivares de arroz, mostramos que a indução de OsNAC5 é maior e antecipada em folhas-bandeira e panículas de plantas da cultivar IR75862, que possui as maiores concentrações de Fe, Zn e proteínas nas sementes. Dessa forma, sugerimos que OsNAC5 é um fator de transcrição do tipo NAC dependente de ABA e associado à senescência, e sua função pode estar relacionada à remobilização de Fe, Zn e aminoácidos dos tecidos verdes para os grãos. Posteriormente, analisamos a expressão de 25 genes de arroz relacionados à homeostase de metais em folhas-bandeira de oito cultivares de arroz (com níveis contrastantes de Fe e Zn nos grãos) durante os estágios de emergência da panícula (R3) e enchimento do grão (R5). Nove destes genes (OsYSL6, OsYSL8, OsYSL14, OsNRAMP1, OsNRAMP7, OsNRAMP8, OsNAS1, OsFRO1 e OsNAC5) apresentaram correlação significativa entre os níveis de expressão em folhas-bandeira e as concentrações de Fe e Zn nas sementes. Assim, nosso estudo forneceu uma pequena lista de possíveis genes-alvo para manipulação da concentração de Fe e Zn em grãos de arroz. / Rice is the staple food for half of the world population. However, it is a poor source of essential micronutrients such as Fe and Zn, most of which are lost during grain processing for food production. For this reason, populations with monotonous diets consisting mainly of rice are especially prone to Fe and Zn deficiency, which are the most common and widespread nutritional disorders in the world, affecting more than 2 billion people. Since flag leaves are one of the sources of remobilized metals for developing seeds, the identification of the molecular players that might contribute to the process of metal transport from flag leaves to the seeds may be useful for biofortification purposes. In this way, we used two different appraches to address this issue. First, we conducted suppression subtractive hybridization (SSH) analysis in flag leaves of two rice cultivars and isolated 78 sequences up-regulated in flag leaves at the grain filling stage (R5) relative to the panicle exertion stage (R3). Differential expression of selected genes (including the OsNAC5 transcription factor) was confirmed by quantitative RT-PCR. We showed that OsNAC5 expression is up-regulated by natural (aging) and induced senescence processes (dark, ABA application, high salinity, cold and Fedeficiency) and its expression is not affected in the presence of 6-benzylaminopurine (a senescence inhibitor) under dark-induced senescence. Salt induction of OsNAC5 expression is abolished by nicotinamide, an inhibitor of ABA effects. This result and the presence of cis-acting elements in the promoter region of the OsNAC5 gene suggest an ABA-dependent regulation. Using four different rice cultivars, we showed that OsNAC5 up-regulation is higher and earlier in flag leaves and panicles of IR75862 plants, which have higher seed concentrations of Fe, Zn and protein. We suggest that OsNAC5 is a novel senescenceassociated ABA-dependent NAC transcription factor and its function could be related to Fe, Zn and amino acids remobilization from green tissues to seeds. We also analyzed the expression of 25 metal-related genes from rice in flag leaves of eight rice cultivars (showing contrasting levels of seed Fe and Zn) during panicle emergence (R3) and grain filling stage (R5). The expression level of nine of these genes (OsYSL6, OsYSL8, OsYSL14, OsNRAMP1, OsNRAMP7, OsNRAMP8, OsNAS1, OsFRO1 and OsNAC5) in flag leaves exhibited significant correlations with Fe and/or Zn concentrations in the seeds. In this way, our study has provided a short list of putative target genes to manipulate Fe and Zn concentrations in rice grains.

Os agrupamentos ferro-enxofre [Fe-S] são grupos prostéticos requeridos em processos essenciais como respiração, fotossíntese, reações metabólicas, sinalização e regulação gênica. Em plantas, a biossíntese das proteínas Fe-S é compartimentalizada e adaptada às necessidades de uma célula eucariótica fotossintetizante. Diversos fatores ambientais afetam o desenvolvimento das plantas e limitam sua produtividade. Dentre esses organismos afetados encontram-se a soja e o arroz, culturas de grande importância comercial no Brasil e no mundo. Foram analisados, pelo método de RT-qPCR, os perfis de expressão dos genes que codificam a enzima cisteína desulfurase NFS1 e NFS2 de ambas as espécies, participantes da rota de formação dos cofatores [Fe-S] mitocondriais e plastídicos, respectivamente. Para Oryza sativa ssp. indica e ssp. japonica, as análises permitiram mostrar uma distribuição diferencial dos transcritos entre órgãos. OsNFS1 apresenta maiores níveis de transcritos tanto em raiz quanto em folha, enquanto OsNFS2 apresentou maiores níveis de transcritos em folha do que em raiz. A resposta destes genes ao frio (24 h a 4ºC) difere entre as subespécies. Na ssp. japonica, os dois genes são induzidos em ambos os tecidos. Para a ssp. indica há redução dos níveis de mRNA de NFS1 e aumento de NFS2 em folhas, e não há respostas em raízes. Nos tratamentos com alumínio na ssp. japonica, há redução dos níveis de mRNA de ambos os genes nas raízes, enquanto não há resposta na parte aérea. As análises em Glycine max, organismo que apresenta duplicação dos genes, permitiram demonstrar que a modulação destes é realizada de maneira diferencial. Em raiz, o tratamento com frio (5 h, 10 h e 24 h a 4ºC) promoveu a redução dos níveis de mRNA de uma das cópias de NFS1 e a indução da outra, enquanto os genes codificadores de NFS2 foram reprimidos. Em folha, o mesmo tratamento induziu uma das cópias de NFS1 e uma de NFS2, enquanto que as outras oscilaram em nível bem inferior de expressão. A exposição de G. max ao ácido salicílico induz a modulação dos genes mitocondriais, aumentando os níveis de mRNA em raízes e reprimindo-os em folhas. As análises dos cis-elementos permitiram mostrar a correlação destes com os dados encontrados em RT-qPCR. Desta maneira, tanto em arroz quanto em soja, a cisteína desulfurase parece ser modulada por diferentes estresses, possuindo padrões diferenciados conforme o órgão e o gene. / Iron-sulfur [Fe-S] clusters are prosthetic groups required to maintain life processes including respiration, photosynthesis, metabolic reactions, sensing, signaling and, gene regulation. In plants the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the eukaryotic and photosynthetic cell. Many environmental factors affect plant development and limit the productivity and the geographical distribution. Among those affected organisms are soybean and rice, crops with huge economic importance worldwide. Here we analyze the expression profile of cysteine desulfurase genes NFS1 and NFS2, involved in the biogenesis of [Fe-S] clusters in mitochondria and plastid, respectively, from both species, by RT-qPCR. Analysis of Oryza sativa ssp. indica and ssp. japonica showed a differential expression between organs, OsNFS1 has a higher expression in roots and leaves than OsNFS2, and OsNFS2 is more expressed in leaves than in roots. Rice subspecies exhibited different responsiveness to cold. The japonica ssp. increased transcript level from both genes in both organs, while ssp. indica decreased OsNFS1 and increased OsNFS2 expressions in leaves, and did not change the expression pattern in roots. Rice ssp. japonica showed a decrease in OsNFS1 and OsNFS2 transcript levels in root, while leaves did not change, under aluminum stress. Soybean analysis, which presents duplication of both genes, demonstrated particular transcript levels considering organ and stress response. GmNFS1 had a high expression in roots, while GmNFS2 did not differ between organs. Cold-treated plants (0, 5, 10 and 24h at 4°C) showed a decrease in cysteine desulfurases transcript level in roots, and an increase in leaves. Plants treated with salicylic acid increased GmNFS1 transcript level in roots, and decreased in leaves. Besides that, an analysis of promoter regions showed the presence of different cis-elements among cysteine desulfurase genes, corroborating with differential expression of each loci. Our results suggest that cysteine desulfurases, in rice and soybean, may be involved in stress response, being modulated by different stimuli according to organ and gene.