Comparative analysis of secondary growth and its related genes between erect and oblique branches of Citrus

Abstract:【Objective】Citrus is an important fruit tree in south China. Because the fruit-bearing shoots of branch are soft, high-yielding trees often require supporting poles to avoid fruit squeezing or branch breaking and to ensure fruit quality and tree health. However, erection of poles needs much labor and it is difficult to finish such work in the current era with labor shortages and aging. Promoting the second-ary growth of branches and simplifying tree structure play an important role in removing erection of poles, reducing labor cost and increasing orchard revenue. The present study is to explore the difference in secondary growth between erect and oblique branches of citrus, explain the reasons behind the differ-ence, and lay a foundation for regulating the secondary growth of citrus shoots.【Methods】Three-year-old red pomelo (Citrus grandis ‘Hongxinyou’) and Nanfang tangerine (C. reticulata ‘Kinokuni’) were used as materials to compare the thickness, length, anatomical structure, and expression of genes related to secondary growth between erect (perpendicular to the ground) and oblique (the shoot is at a 45-60 de-gree angle to the ground level) shoots.【Results】The thickness of the erect shoots of Nanfeng tangerine and red pomelo was significantly higher than that of the oblique shoots. Moreover, the length of the erect shoots of the two varieties was slightly longer than that of the oblique shoots, but the difference was not significant. The anatomical analysis showed that the primary phloem cell size was larger, the cambium width was significantly larger, and the xylem width was significantly lower in the upper part of erect shoots than in that of the oblique shoots, while in the lower part of shoots, the width of cambi-um and xylem of the erect shoots was significantly higher than that of oblique shoots. The expression levels of 34 genes related to secondary growth were compared. Of the four genes related to xylem sec-ondary growth, KNAT1 and STM in the erect shoots of Red pomelo were significantly more active than those in the oblique shoots, but there was no significant difference between them in Nanfeng tangerine; moreover in both varieties, the expression level of SND1 in the erect shoots was significantly higher than that in the oblique shoots, but the expression level of BOP1/2 was not significantly different be-tween the two types of shoots. The expression levels of APL, BRX, BRXL2, NEN1/2- 1, NEN4 and BAM3 related to phloem secondary growth were significantly lower and the expression levels of BRXL4 and NAC45/86 were significantly higher in red pomelo the erect shoots than in the oblique shoots; in Nanfeng tangerine erect shoots, the expression levels of APL, BRXL2, BRXL4, NAC45/86 and NEN1/2-1 were significantly lower and the expression levels of NEN4, OPS and BAM3 were significantly higher than those of the oblique shoots. As for the 15 genes related to secondary growth in cambium, the ex-pression levels of WOX4, WOX14, MYB46-1, MYB46-2, PXY, AF1 and NST1 were significantly higher and the expression levels of BES1, CLE8, HAM4 and NAC3 were significantly lower the erect shoots than in the oblique shoots in red pomelo; in the erect shoots of Nanfeng tangerine, the expression levels of WOX4, CLE8, MYB46- 1, and PXY were significantly higher and the expression levels of BES1, WOX14, HAM4, KNAT6, MYB46- 2, MYB59, and SD1 were significantly lower than in the oblique shoots. Finally, the expression levels of two genes (ARR11 and ARR12) related to cytokinin synthesis and three genes (ARF5, ATHB8 and PIN1) related to auxin transport were also compared between the two types of shoots in both varieties. The expression level of ARR12 in Nanfeng tangerine was signifi-cantly higher in the erect shoots than in the oblique shoots; the expression level of ARF5 in red pomelo was significantly higher and the expression level of PIN1 was significantly lower in the erect shoots than in the oblique shoots; moreover, the expression level of ATHB8 was significantly lower in erect shoots in both varieties.【Conclusion】The difference in cambium activity and xylem thickness was the main reason for the difference in shoot thickening in citrus. Specially, PXY and WOX4 played a key role in the maintenance of cambium activity. The expression level of the two genes may be the key reason to determine the speed of shoot secondary growth. On the other hand, the high expression of SND1 in erect branches of citrus may be the important reason for their thicker (wider) xylem than in the oblique branches. Namely, the high expression of cambium-related genes, WOX4 and PXY, and xylem-related gene SND1 may contribute to the higher shoot thickening growth of erect shoots. The function of such genes needs to be further verified.