Karakterisasi Lignin dan Agromorfologi Padi Indonesia Serta Analisis Ekspresi OsCAD2 dan OsMYB55/61 Terkait Biosintesis Lignin

Abstract

Padi (Oryza Sativa L.) merupakan anggota Gramineae yang berpotensi tinggi untuk dimanfaatkan sebagai sumber biomassa lignoselulosa. Manipulasi kandungan lignin menjadi bahan kimia aromatik yang lebih bermanfaat telah dikembangkan sebagai salah satu strategi pengembangan biomassa lignoselulosa. Informasi mengenai kandungan lignin dan gen yang berkaitan dengan biosintesis lignin merupakan hal yang penting namun masih sangat terbatas pada padi Indonesia. Tujuan penelitian ini adalah untuk menganalisis kandungan lignin total tiga puluh aksesi padi Indonesia, menganalisis komposisi lignin, membuktikan korelasi antara karakter kandungan lignin total terhadap tujuh karakter agromorfologi terkait lignin, membandingkan pola ekspresi dua gen terkait biosintesis lignin (OsCAD2 dan OsMYB55/61) pada aksesi padi terpilih dengan qPCR, dan menganalisis sifat mekanik batang (flexural strength dan tensile strength) terkait kandungan lignin total pada aksesi padi terpilih. Kandungan lignin total dianalisis menggunakan metode Thioglycolic Acid Lignin (TGAL), karakterisasi monomer lignin menggunakan Py-GC-MS (Pyrolysis-GC-MS), dan uji mekanik batang dilakukan menggunakan Universal Testing Machine. Hasil penelitian menunjukkan rentang variasi kandungan lignin total batang aksesi padi berkisar 8,38–20,75% CWR (Cell Wall Residue). Tiga aksesi kandungan lignin tertinggi (Inpago 7, Lampung Kuning, dan Gebang) dan tiga aksesi kandungan lignin total terendah (Tukad Petanu, Ciherang, dan Cigenit) telah teridentifikasi. Hasil pengamatan menunjukkan kandungan lignin total pada padi berkorelasi positif signifikan dengan karakter tinggi tanaman, panjang batang, panjang malai, diameter batang, bobot total segar dan bobot malai per anakan. Sebaliknya, karakter jumlah anakan berkorelasi negatif signifikan dengan kandungan lignin. Hasil analisis qPCR terhadap enam aksesi padi terpilih menunjukkan bahwa pola ekspresi OsCAD2 konsisten terhadap kandungan lignin total dan secara histokimia dengan pewarnaan Phloroglucinol HCl pada heading stage menunjukkan deposisi lignin di vascular bundle dan cortical fiber, menyatakan bahwa OsCAD2 berperan penting dalam biosintesis lignin padi. Sebaliknya, OsMYB55/61 menunjukkan pola yang tidak konsisten terhadap kandungan lignin total, diduga karena tidak hanya berperan dalam regulasi biosintesis lignin namun juga berperan dalam biosintesis selulosa. Proporsi monomer siringil (S) dan rasio S/G pada aksesi padi kandungan lignin total tinggi lebih tinggi dibandingkan aksesi padi kandungan lignin total rendah, sehingga kemungkinan lebih mudahdihidrolisis saat proses delignifikasi. Hasil uji kekuatan mekanik mengindikasikanbahwa sifat mekanik batang dipengaruhi oleh kandungan lignin total dan kemungkinan berpengaruh positif terhadap sifat resistensi kerebahan pada padi.

Rice (Oryza sativa L.), belongs to the family of Gramineae, has high potential as a source of lignocellulosic biomass. Manipulating lignin content into more useful aromatic chemicals has been widely developed as one of the strategies for developing lignocellulosic biomass. Information about lignin content and genes related to lignin biosynthesis in Indonesian rice accessions are essential; however it is still very limited. This study aimed to analyze the lignin contents of thirty Indonesian rice accessions, analyze the lignin composition of selected high and low lignin contents rice accession, study the correlation between lignin content and lignin-related to seven agromorphological characters, compare the expression patterns of two predicted lignin biosynthetic genes (OsCAD2 and OsMYB55/61) of selected rice accessions by qPCR, and analyze the mechanical properties of culms (flexural strength and tensile strength) related to lignin contents in the selected rice accessions. Total lignin content was analyzed using the Thioglycolic Acid Lignin (TGAL) method, while the lignin monomers were characterized by Py-GC-MS (Pyrolysis-GC-MS), and the mechanical properties of culm were tested by the Universal Testing Machine. The TGAL results showed that the total lignin contents of the rice accessions ranged from 8.38 to 20.75% CWR (Cell Wall Residue). Three accessions with the highest (Inpago 7, Lampung Kuning, and Gebang) and three accessions with the lowest (Tukad Petanu, Ciherang, and Cigenit) total lignin contents were identified. The results also showed that total lignin content in rice was significantly positively correlated with the characteristics of plant height, stem length, panicle length, stem diameter, total fresh weight, and panicle weight per tiller. On the other hand, the number of tillers was significantly negatively correlated with lignin content. qPCR analysis of the six selected rice accessions showed that OsCAD2 expression patterns at the heading stages were consistent with their total lignin contents, which was also shown by the lignin deposition in vascular bundle and cortical fiber as analyzed by Phloroglucinol HCl staining at the heading stage, suggesting its important roles in rice lignin biosynthesis. In contrast, OsMYB55/61 showed inconsistent expression patterns to total lignin contents, which may also upregulate not only lignin biosynthesis but also cellulose biosynthesis. The proportion of syringyl monomer (S) and the ratio of S/G in rice accessions with high total lignin content was higher than in accessions with low lignin content, thus it might be easier to be hydrolyzed in delignification process. The results of the mechanical strength test indicated the mechanical properties of the culm were affected by the total lignin content and might positively affect lodging resistance in rice.